CN110944665B - AMHRII-binding compounds for the prevention or treatment of lung cancer - Google Patents

Abstract

The present invention relates to the use of a human AMHRII binding agent for the prevention or treatment of lung cancer, in particular to the use of a human AMHRII binding agent for the prevention or treatment of non-small cell lung cancer (NSCLC), even more particularly to the use of a human AMHRII binding agent for the prevention or treatment of NSCLC selected from the group comprising: epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC, and neuroendocrine NSCLC.

Description

AMHRII-binding compounds for the prevention or treatment of lung cancer

Technical Field

The invention relates to the field of lung cancer treatment.

Background

Lung cancer is a malignant transformation and expansion of lung tissue and results in 130 tens of thousands of deaths annually. It is the most common cause of cancer-related death in men and the second most common cause in women.

The world health organization (World Health Organization) classifies lung cancer into four major histological types: (1) Squamous Cell Carcinoma (SCC), (2) adenocarcinoma, (3) large cell carcinoma, and (4) Small Cell Lung Carcinoma (SCLC). The term non-small cell lung cancer (NSCLC) includes squamous carcinoma, adenocarcinoma, and large cell carcinoma.

Non-small cell lung cancer (NSCLC) falls into a class because of their prognosis and method of treatment are approximately the same. There are mainly three subtypes: squamous cell lung carcinoma, adenocarcinoma, and large cell lung carcinoma. Squamous cell carcinoma accounts for 29% of lung cancer, and also begins with the larger bronchi, but grows slower. The size of these tumors varies from diagnosis to diagnosis. Adenocarcinomas are the most common subtype of NSCLC, accounting for 32% of lung cancers. It is in a form that starts near the gas exchange surface of the lung. Most cases of adenocarcinoma are associated with smoking. However, among never-smoked people ("never-smoked people"), adenocarcinoma is the most common form of lung cancer. Bronchioloalveolar cancer is a subtype of adenocarcinoma, more common in women from non-smokers, and may have a different response to treatment. Other subtypes of NSCLC are neuroendocrine lung cancer (NE), acinar lung cancer (AT), and large cell carcinoma (fast-growing form), accounting for 9% of lung cancers that grow near the lung surface.

Small cell lung cancer (SCLC, also known as "oat cell carcinoma") is a less common form of lung cancer. It tends to start in a larger breathing tube and grow rapidly to a large size. The most commonly involved oncogene is L-myc. "oat" cells contain dense neurosecretory particles that allow for endocrine/paraneoplastic syndrome association. It is initially more sensitive to chemotherapy, but ultimately has a poorer prognosis and is often shifted when it occurs. This type of lung cancer is closely related to smoking.

Other types of lung cancer include carcinoid, adenoid cystic carcinoma (cylindrical tumors) and myxoepidermoid carcinoma.

Early detection is difficult because clinical symptoms usually do not appear until the disease is advanced. Currently, diagnosis is aided by using chest x-rays, analyzing the type of cells contained in saliva, and fiber optic examination of bronchial passages. The treatment regimen is determined by the type and stage of the cancer and includes surgery, radiation therapy, and/or chemotherapy. Although there has been much research into therapies for this disease, lung cancer remains difficult to treat.

Known lung cancer treatments include surgery, chemotherapy, radiation therapy, and targeted drug therapies.

Targeted therapies, particularly targeted immunotherapy, may benefit lung cancer patients for whom more conventional chemotherapy or radiation therapy is ineffective. Targeted immunotherapy involves the use of monoclonal antibodies.

The monoclonal antibodies bevacizumab (anti-VEGF antibody) and ramucirumab (anti-VEGFR 2 antibody) aim to prevent tumor neovasculature, whereas rituximab (anti-EGFR) targets growth by preventing the action of another growth factor. Currently, at least two immune checkpoint inhibitor (pembrolizumab/anti-PD 1 and nivolumab/anti-PD 1) targeting antibodies against lung cancer patients have been approved. Today, long term remissions and longer survival rates can be obtained using such immune-based therapies (such as checkpoint inhibitors, monoclonal antibodies, therapeutic vaccines, and adoptive cell therapies).

However, there remains a need in the art for additional tools for lung cancer therapies that may be an alternative or complement to existing therapies.

Disclosure of Invention

The present invention relates to the use of human AMHRII binding agents for the prevention or treatment of lung cancer. The invention then relates to the use of a human AMHRII binding agent in a method for preventing or treating lung cancer in a patient suffering from lung cancer.

The lung cancer may be selected from the group comprising non-small cell lung cancer (NSCLC), in particular NSCLC selected from the group comprising: epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCLC, polymorphic cell carcinoma NSCLC and neuroendocrine NSCLC.

In a preferred embodiment, the human AMHRII binding agent is used to treat the lung cancer specified above that expresses AMHRII at a sufficient level of expression at the cell membrane.

In a most preferred embodiment, the sufficient level of expression is expressed as a threshold AMHRII expression score value detailed elsewhere in this specification.

In some embodiments, the human AMHRII binding agent consists of an anti-AMHRII monoclonal antibody.

In some embodiments, the human AMHRII binding agent consists of an Antibody Drug Conjugate (ADC).

In some embodiments, the human AMHRII binding agent consists of AMHRII binding to an engineered receptor.

In some embodiments, the human AMHRII binding agent consists of cells that express AMHRII that bind to an engineered receptor (such as CAR T cells or NK T cells that express AMHRII that bind to an engineered receptor).

In some embodiments, the AMHRII binding agent is combined with one or more different anticancer agents.

The invention also relates to a method for determining whether an individual is suitable for lung cancer treatment with a AMHRII binding agent as defined above, wherein the method comprises the step of determining whether a lung tumour tissue sample previously obtained from the individual expresses AMHRII protein at the cell surface.

The present invention relates to a method for determining whether an individual is responsive to lung cancer treatment with AMHRII binding agents as defined above, wherein the method comprises the step of determining whether a lung tumor tissue sample previously obtained from the individual expresses AMHRII protein at the cell surface.

Drawings

Fig. 1 shows amino acid sequences of VH and VL domains of multiple variants of the 3C23 monoclonal antibody. Figure 1A shows VH domains for each antibody variant. FIG. 1B shows the VL domain of each antibody variant.

FIG. 2 shows AMHRII expression of various cancer cell lines.

FIG. 2A shows AMHRII MRNA expression of a cancer cell line. Abscissa: from left to right in fig. 2A: HCT116 (colorectal carcinoma), COV434-WT (human ovarian granulosa cell tumor), K562 (human myelogenous leukemia) and OV90 (human malignant papillary serous adenocarcinoma). Ordinate: AMHRII MRNA expression levels, as determined by RT-qPCR, are expressed in arbitrary units (RQ).

Fig. 2B to 2F: AMHRII protein membrane expression by the same cancer cell line as in fig. 2A: HCT116 (FIG. 2B), COV434-WT (FIG. 2C), K562 (FIG. 2D), NCI-H295R (FIG. 2E) and OV90 (FIG. 2F). Abscissa: fluorescent signal intensity in arbitrary units (FL 2-A dye). Ordinate: cell count.

FIG. 3 shows AMHRII expression of various lung cancer cells as measured by flow cytometry.

FIG. 3A shows AMHRII protein membrane expression from cells of xenografts from squamous cell lung carcinoma patients (Ref Lu 7860). FIG. 3B shows AMHRII protein membrane expression from cells from xenografts derived from patients with lung cancer (Ref Lu 7166). FIG. 3C shows AMHRII protein membrane expression of cells from xenografts from squamous cell lung carcinoma patients (Ref Lu 7298). FIG. 3D shows AMHRII protein membrane expression of cells from xenografts from squamous cell lung carcinoma patients (Ref Lu 7414). FIG. 3E shows AMHRII protein membrane expression of cells from a xenograft derived from a patient with polymorphous cell lung cancer (Ref Lu 7558). Abscissa: fluorescence intensity (FL 2-A dye) is expressed in arbitrary units. Ordinate: cell count.

Figure 3F shows AMHRII protein membrane expression from cells from healthy edges of surgically resected human NSCLC (whose FACS curve is shown in figure 3G).

Figure 3G shows AMHRII protein membrane expression from cells of a surgically excised fresh sample of human NSCLC.

In fig. 3: (i) left peak: cells incubated with an unrelated isotype antibody; (ii) right peak: cells incubated with 3C23K anti-AMHRII antibody.

Abscissa: fluorescence intensity (FL 2-A dye) is expressed in arbitrary units. Ordinate: cell count.

Figure 4 shows the relative weight change of animals xenografted with human lung cancer cells. Treatment began on day 18 after SC131 implantation. The vehicle and GM102 were administered at 20mg/kg i.v. once every two weeks for 3 weeks. At D0, 20mg/kg of docetaxel was administered slowly i.v. once. 5mg/kg cisplatin and 100mg/kg gemcitabine are administered i.p. once a week for 1 to 3 weeks. Initial group size: 9 animals. Ordinate: relative body weight, expressed in kg (mean +/-mean standard error (sem)). Abscissa: ● A vehicle; ■ GM102 20mg/kg; 20mg/kg of docetaxel; is a combination of GM102 and docetaxel; a combination of cisplatin 5mg/kg and gemcitabine 100 mg/kg; GM102, cisplatin, and gemcitabine.

Fig. 5 shows tumor growth changes induced by 3C23K anti-AMHRII antibodies in combination with other anti-cancer agents or without other anti-cancer agents in animals xenografted with human lung cancer cells. Treatment was started on day 18 after SC131 implantation. The vehicle and GM102 were administered at 20mg/kg i.v. once every two weeks for 3 weeks. At D0, 20mg/kg of docetaxel was administered slowly i.v. once. Cisplatin 5mg/kg and gemcitabine 100mg/kg are administered i.p. once a week for 1 to 3 weeks. Initial group size: 9 animals. Ordinate: tumor volume, expressed in mm ³ (mean +/-sems). Abscissa: ● A vehicle; ■ GM102 20mg/kg; 20mg/kg of docetaxel; is a combination of GM102 and docetaxel; a combination of cisplatin 5mg/kg and gemcitabine 100 mg/kg; GM102, cisplatin, and gemcitabine.

FIG. 6 shows the anti-tumor activity of 3C23K anti-AMHRII antibodies in combination with other anti-cancer agents or without other anti-cancer agents on animals xenografted with human lung cancer cells. Treatment was started on day 18 after SC131 implantation. The vehicle and GM102 were administered at 20mg/kg i.v. once every two weeks for 3 weeks. At D0, 20mg/kg of docetaxel was administered slowly i.v. once. Cisplatin 5mg/kg and gemcitabine 100mg/kg are administered i.p. once a week for 1 to 3 weeks. Initial group size: 9 animals. Ordinate: such as TC in percent. Abscissa: ● A vehicle; ■ GM102 20mg/kg; 20mg/kg of docetaxel; is a combination of GM102 and docetaxel; a combination of cisplatin 5mg/kg and gemcitabine 100 mg/kg; GM102, cisplatin, and gemcitabine.

Figure 7 shows the tumor growth changes induced by GM102 (low fucose anti AMHRII antibody) in animals xenografted with squamous non-small cell lung cancer tumor xenografts. Each dashed curve: xenograft animals administered with vehicle solution. Each continuous curve: xenograft animals administered GM 102. Abscissa: the period of time after initiation of treatment is expressed in days. Ordinate: tumor volume, expressed in mm ³.

Figure 8 shows GM102 (low fucose anti AMHRII antibody) -induced tumor growth changes on animals xenografted with squamous non-small cell lung cancer tumor xenografts at day 28 post treatment initiation. Fig. 8A and 8B, ordinate: tumor volume, expressed in mm ³. Fig. 8A and 8B, abscissa: (i) left side: xenograft animals administered with vehicle solution; (ii) right side: xenograft animals administered GM 102. Fig. 8A: absolute results of each xenograft animal tested. Fig. 8B: mean +/-standard deviation calculated from the results depicted in fig. 8A.

Detailed Description

The inventors have surprisingly shown that AMHRII receptors are expressed at the cell membrane of non-small cell lung cancer tissue, in particular at the cell membrane of epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, polymorphous cell carcinoma NSCLC, squamous cell carcinoma NSCLC and neuroendocrine NSCLC subtypes. In contrast, no membrane level AMHRII was detected in SCLC or NSCLC from the neuroendocrine subtype or acinar subtype.

The term "AMHR-II" represents a human anti-Mueller hormone type II receptor. The sequence of human AMHR-II is described herein as SEQ ID NO.18 (lacking signal peptide MLGSLGLWALLPTAVEA (SEQ ID NO: 17)).

As used herein, the term "PDX" is an acronym for the expression "patient-derived xenograft". Patient-derived xenografts are highly used in vivo models of cancer in which tissues or cells from a patient's tumor are implanted (i.e., "transplanted") into an immunodeficient non-human mammal (e.g., an immunodeficient mouse).

As shown in the examples herein, the inventors found AMHRII to be expressed at the cell membrane of lung cancer tissue at a variable frequency depending on the subtype of lung cancer considered.

To the inventors' knowledge, the membrane expression of AMHRII in lung cancer cells is shown for the first time herein.

Illustratively, as shown in the examples herein, AMHRII is expressed more frequently by cancer cells derived from tumor tissue derived from a patient having epidermoid NSCLC lung cancer or adenocarcinoma NSCLC large cell NSCLC lung cancer than by cancer cells derived from tumor tissue derived from a patient having squamous NSCLC or large cell NSCLC. The detection of a relatively high frequency means that cancer patients with one of these four lung cancers are more frequently eligible for targeting AMHRII of the anti-cancer therapies (i.e., more frequently responsive to AMHRII-targeted anti-cancer therapies), but such anti-cancer therapies are less frequently associated with treating patients with neuroendocrine NSCLC.

As shown in the examples herein, any NSCLC lung cancer can be treated with AMHRII binding agents, provided that tumor cells from the non-gynecological tumor express AMHRII at their membrane, and thus provided that the presence of AMHRII protein at the tumor cell membrane can be detected or determined according to any method.

Thus, the experimental data provided in the examples herein demonstrate that the same AMHRII binding agent (here, an anti-AMHRII monoclonal antibody) is effective for treating a variety of different NSCLC lung cancers, provided that the AMHRII target protein is expressed at the tumor cell membrane.

Incidentally, in the field of anticancer active ingredients composed of target binding molecules (e.g., target binding antibodies), the case where the same active ingredient is effective for treating a plurality of different cancers is not unprecedented. Illustratively, an anti-PD 1 antibody named pembrolizumab has been licensed by the U.S. Food and Drug Administration (FDA) as an active ingredient that can be used to treat a variety of different types of cancers, provided that the cancers share the same physiological characteristics.

As used herein, expression AMHRII at the cell membrane of a lung cancer cell means that the lung cancer cell expresses AMHRII at or above a given quantifiable level.

According to some embodiments, responsiveness of an individual with lung cancer to AMHRII binding molecule therapy may be assessed by determining whether lung cancer cells from a sample previously collected from the individual are expressing AMHRII at their membrane.

According to some embodiments, responsiveness of an individual with lung cancer to AMHRII binding molecule therapy may be assessed by determining whether lung cancer cells from a sample previously collected from the individual are above a determined threshold expression AMHRII at their membrane.

The AMHRII membrane expression level that can be used in some embodiments to determine responsiveness of a patient with non-gynaecological cancer to treatment with AMHRII binding agents (e.g., anti-AMHRII antibodies) can be assessed using a variety of techniques including (i) the percentage of tumor cells contained in a tumor sample that expresses AMHRII at its membrane, (ii) the average number of AMHRII proteins at the tumor cell membrane, and (iii) the FACS AMHRII signal profile of tumor cells contained in a tumor cell sample that is tested.

According to some embodiments, when membrane AMHRII% or more of lung tumor cells contained in a tumor sample previously collected from an individual having lung cancer are detected, lung cancer cells contained in the tumor sample may be assessed as expressing membrane AMHRII.

Thus, in some embodiments, an individual with lung cancer is determined to be responsive to AMHRII binding agent treatment when 5% or more of the lung tumor cells contained in a tumor sample previously collected from the individual are expressed AMHRII at their membrane.

Methods for determining the frequency (e.g., percent) of tumor cells expressing membrane AMHRII protein are disclosed elsewhere in this specification, including in the examples herein.

According to some embodiments, responsiveness of a patient with lung cancer to a cancer treatment of AMHRII binding agent (e.g., an anti-AMHRII antibody) may be assessed by determining the average amount of AMHRII protein present at the tumor cell membrane contained in a tumor sample previously collected from the patient.

In some embodiments, a patient with lung cancer may be classified as responsive to AMHRII binding agent therapy, e.g., anti-AMHRII antibody therapy, when the average number of membrane AMHRII proteins expressed by tumor cells contained in a tumor sample previously collected from the patient has 10000 AMHRII proteins or more.

Assessment of the amount of AMHRII protein expressed at the lung tumor cell membrane can be performed by using conventional methods including: (a) A step of incubating a sample containing cells from a tumor tissue sample previously collected from a patient with a detectable compound that specifically binds to AMHRII protein (such as a fluorescently labeled anti-AMHRII antibody), and additionally (b) a step of determining the amount of the detectable compound (e.g., the amount of a fluorescently labeled anti-AMHRII antibody) bound to each test cell from the sample. As shown in the examples herein, assessing the amount of AMHRII protein expressed at the tumor cell membrane may be performed, for example, by using well known Fluorescence Activated Cell Sorting (FACS) techniques.

In still other embodiments, a patient with lung cancer may be classified as responsive to AMHRII binding agent treatment, e.g., as responsive to anti-AMHRII antibody treatment, by analyzing AMHRII FACS curves of tumor cells contained in tumor samples previously collected from the patient.

According to these other embodiments, in a Fluorescence Activated Cell Sorting (FACS) method, a patient with lung cancer may be classified as responsive to AMHRII binder treatment, e.g., as responsive to anti-AMHRII antibody treatment, when the ratio of (i) the Mean Fluorescence Intensity (MFI) value obtained from tumor cells incubated with an isotype fluorescent-labeled antibody to (ii) the mean fluorescence intensity of tumor cells incubated with an anti-AMHRII fluorescent-labeled antibody is 1.5 or greater.

To determine the average fluorescence intensity ratio, both isotype antibodies and anti-AMHRII antibodies were labeled with the same fluorescent agent, such as alexa fluor 488 dye sold by sameir feier science company (Company ThermoFisher Scientific), as shown in the examples herein.

In some further embodiments, responsiveness of a lung cancer individual to AMHRII binding agent treatment can be determined by calculating a AMHRII expression score that allows differentiation between (i) lung cancer cells that are derived from the expression membrane AMHRII of lung cancer that can be treated with AMHRII binding agent and (ii) lung cancer cells that are derived from the expression membrane AMHRII of lung cancer that cannot be treated with AMHRII binding agent.

Thus, the inventors have determined that patients with lung cancer that are particularly suitable for cancer treatment using the AMHRII binding agents described herein (i.e., particularly those with lung cancer that are responsive to cancer treatment using the AMHRII binding agents described herein) encompass those patients with cancer tumors that express AMHRII at the cell membrane at a level sufficient for constituting the relevant cellular targets to be destroyed.

Then, according to these further embodiments, the inventors have determined that the minimum AMHRII expression level measured in a cancer cell sample from a lung cancer patient can confirm that the patient is responsive to treatment with a AMHRII binding agent, and thus can be treated by a AMHRII binding agent as described herein.

Thus, when assessing the level of AMHRII expression of lung cancer cells contained in a sample previously collected from an individual having lung cancer by determining (i) the frequency of tumor cells expressing membrane AMHRII (e.g., the percentage of tumor cells expressing AMHRII at their membrane) and (ii) the level of AMHRII membrane expression of the tumor cells (e.g., the average number of membrane AMHRII proteins per cell), the responsiveness of the individual to treatment with AMHRII binding agent can also be determined.

Thus, in some of these additional embodiments, the inventors determined that responsiveness of a lung cancer patient to a human AMHRII binding agent, e.g., to an antibody against human AMHRII, is desirable in a sample of tumor cells previously collected from the patient if (i) the tumor cells contained in the sample have a minimum average number of human AMHRII proteins at their membranes and (ii) the frequency of cells expressing human AMHRII at their membranes (e.g., the percentage of cells expressing human AMHRII at their membranes) is at least a threshold.

Thus, another method is also described herein that can also be used to determine a particular AMHRII expression score value to allow differentiation between (i) lung cancer patients unsuitable for cancer therapy with a AMHRII binding agent (i.e., lung cancer patients that do not respond to cancer therapy with a AMHRII binding agent) and (ii) lung cancer patients suitable for cancer therapy with a AMHRII binding agent (i.e., lung cancer patients that respond to cancer therapy with a AMHRII binding agent (e.g., an anti-human AMHRII antibody).

More precisely, according to embodiments of the above methods, patients suffering from lung cancer as described herein and treatable with AMHRII binding agents as described herein for lung cancer are preferably those patients whose membrane AMHRII expression score value has been determined to be 1.0 or greater.

The membrane AMHRII expression score may be based on an immunohistochemical evaluation of AMHRII expression of the lung cancer cells tested and is an average of the membrane AMHRII scores determined from a plurality of lung cancer cell samples derived from different individuals with lung cancer, and wherein (i) the individual membrane AMHRII score for a given lung cancer cell sample is designated 0 if no AMHRII expression is detected, (ii) the individual membrane AMHRII score for a given lung cancer cell sample is designated 1 if significant AMHRII expression is detected, (iii) the individual membrane AMHRII score for a given lung cancer cell sample is designated 2 if high AMHRII expression is detected, and (iv) the individual membrane AMHRII score for a given lung cancer cell sample is designated 3 if overexpression of AMHRII is detected.

In fact, there was a relationship between (i) the score assigned to the expression level of membrane AMHRII by immunohistochemical evaluation as described above and (ii) the average number of AMHRII proteins expressed per lung cancer cell. In the examples herein, it is shown that, starting from a sample of lung tumor cells previously collected from a lung cancer patient, by determining the average number of membrane AMHRII proteins per cell, it is also possible to evaluate the membrane AMHRII expression level that enables the designation of individual membrane AMHRII scores.

According to the above embodiments of determining responsiveness of an individual with lung cancer to treatment with AMHRII binding agent (i.e., to treatment with an anti-AMHRII antibody), for a given lung cancer cell sample, the membrane AMHRII expression score is determined by considering both: (i) The frequency of AMHRII expressing cells in the lung cancer cell sample and (ii) the level of membrane AMHRII expression of the AMHRII expressing cells. In general, the membrane AMHRII expression score for a given lung cancer cell sample is determined by the following formula (I):

e score = frequency x AMHRII _level, where

The E score means a membrane AMHRII expression score value for a given lung cancer cell sample,

Frequency means the frequency of cells contained in the lung cancer cell sample directed to detection of membrane AMHRII expression, and

-AMHRII _level means the level of AMHRII membrane expression of AMHRII expressing cells contained in the given lung cancer cell sample.

Illustratively, the E score for a given lung cancer cell sample is determined to be 1.0, where (i) 50% of the cells express AMHRII (frequency value 0.5), and (ii) AMHRII expression level (AMHRII _level) is 2.

In some embodiments, AMHRII expression scores (or E scores) are determined by immunohistological methods as shown in the examples herein. According to these preferred embodiments, AMHRII membrane expression is assessed by using a detectable antibody specific for AMHRII and by (i) determining the frequency of cells bound by the anti-AMHRII antibody and (ii) determining the intensity of the signal generated by the detectable anti-AMHRII antibody after binding to AMHRII of membrane expression.

Although, as shown in the examples herein, lung cancer cells expressing AMHRII have been determined with a membrane AMHRII expression score of 1.0 or greater for various lung cancers, albeit at different frequencies.

To determine the level of AMHRII membrane expression, detection of AMHRII at the cell membrane should most preferably be performed by using an anti-AMHRII monoclonal antibody with high affinity and high specificity for AMHRII, which anti-AMHRII monoclonal antibody is illustrated in the example by a 3C23K anti-AMHRII monoclonal antibody.

Furthermore, in view of determining AMHRII scores, determining AMHRII expression by immunohistochemical methods most preferably involves careful pretreatment of the lung tissue sample prior to contacting the sample with an appropriate detection reagent (e.g., a high affinity anti AMHRII monoclonal antibody, such as monoclonal 3C23K antibody, having a Kd value of 55.3pM for binding to AMHRII). Sample pretreatment should allow for increased utilization of the AMHRII molecules of detection reagent expressed at the cell surface. Illustratively, as shown in the examples herein, the pre-processing method may include an appropriate combination of specific steps, such as: (i) A system for dewaxing by exposure to a microwave source and (ii) for amplifying a signal generated by the binding of AMHRII binding reagents (such as biotinylated anti-AMHRII antibodies) that can be subsequently complexed with streptavidin conjugated detectable reagents. The pretreatment dewaxing step appears to be important for reversing the detection signal extinction effect due to the previous tissue fixation step. The inventors have shown that AMHRII detectability is particularly sensitive to the effects of formalin used in the tissue fixation step.

This means that although AMHRII binders may be relevant therapeutic agents for treating patients with lung cancer, it is preferred to test AMHRII expression of tumor-derived lung cancer cells first to determine that a particular patient will be administered a AMHRII binder as described herein.

Furthermore, the inventors have shown that anti-AMHRII antibodies can be advantageously used in the treatment of lung cancer.

Thus, the inventors herein have shown that agents targeting AMHRII are useful as novel therapeutic tools for preventing or treating these kinds of cancers, in particular NSCLC selected from the group comprising: epidermoid NSCLC, polymorphic cell carcinoma NSCLC and neuroendocrine NSCLC adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC and neuroendocrine NSCLC.

According to the present invention, the expression "comprising" such as in "comprising the following steps" is also understood as "consisting of, such as" consisting of the following steps ".

The AMH receptor (AMHR or AMHR2 or AMHRII) is a serine/threonine kinase with a single transmembrane domain, belonging to the class II receptor family of TGF- β related proteins. Type II receptors bind to the ligand themselves, but the presence of type I receptors is required for signal transduction. Imbeaud et al (1995,Nature Genet, vol.11:382-388) cloned the human AMH type II receptor gene. Human AMH receptor protein consists of 573 amino acids: 17, 127, 26 and 403 of these 573 amino acids form the signal sequence, extracellular domain (ECD), transmembrane domain and intracellular domain (containing serine/threonine kinase domain), respectively.

As used herein, the term "AMHRII" refers to a human anti-mullerian hormone type II receptor having the amino acid sequence of SEQ ID No. 17.

Expression of anti-mullerian hormone receptor (AMHRII) has been described in the art in gynaecological cancers (i.e., tumors that are mostly infiltrated with immune myeloid cells). AMHRII have been identified as target molecules for the treatment of gynaecological cancers. Antibodies to AMHRII have been raised as therapeutic tools for the treatment of these cancers. Particular mention may be made of the 12G4 anti-AMHRII antibody and variants thereof described in PCT applications No. wo 2008/053330 and No. wo 2011/141653 for the treatment of ovarian cancer, and the 3C23K anti-AMHRII antibody described in PCT applications. PCT application No. wo 2017/025458 may also be mentioned, which discloses specific therapeutic strategies for ovarian cancer by using anti-AMHRII antibody drug conjugates.

Beck et al (2016,Cell Reports, volume 16: 657-671) also describe the expression of the anti-Mueller hormone receptor gene (AMHRII gene). These authors indicate that AMH signaling is an important factor in epithelial plasticity, survival signaling, and selective drug resistance in NSCLC. The work of Beck et al (2016) provides insight into the intracellular mechanisms of NSCLC pathogenesis, particularly by reporting that the identification and characterization of the previously undefined autocrine signaling axes (involving anti-muller hormone and its type II receptor) in NSCLC tumor subgroups by using siRNA to regulate various gene expressions of interest is critical for the response of the Hsp90 inhibitor, genitinib and the approved chemotherapy cisplatin. These authors also found by western blot experiments that the low abundance of AMH proteins and AMHR2 proteins present in the cells of the three cell lines (i.e. a549 and H1299) were blocked by SiRNA targeting the corresponding genes.

The inventors have now unexpectedly found that AMHRII is also expressed at the surface of various human lung cancer cells, including particularly non-small cell lung cancer (NSCLC) cells, even more particularly NSCLC selected from the group comprising: epidermoid NSCLC, polymorphic cell carcinoma NSCLC and neuroendocrine NSCLC adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC and neuroendocrine NSCLC. The inventors also showed that there is no relationship between (i) AMHRII gene expression in cancer cells and (ii) cell membrane AMHRII protein expression in the same cancer cells.

The inventors' findings regarding AMHRII surface expression of human lung cancer cells come from, inter alia, immunohistochemical tests using anti-AMHRII antibodies, performed using human lung tumor tissue samples previously obtained from lung cancer patients. The inventors' findings regarding AMHRII surface expression of human lung cancer cells were also obtained from immunohistochemical tests with anti-AMHRII antibodies, performed in mice on lung tumor tissue samples derived from human primary lung cancer cell xenografts.

The inventors also show that anti-AMHRII antibodies are useful in the treatment of human lung cancer expressing AMHRII at the surface of tumor cells, particularly those expressing AMHRII disclosed in the present specification, including non-small cell lung cancer, particularly epidermoid NSCLC, polymorphic and neuroendocrine NSCLC adenocarcinoma NSCLC, large and squamous cell carcinoma NSCLC, and neuroendocrine NSCLC. Notably, anti-AMHRII antibodies as well as anti-AMHRII antibodies in combination with chemical anticancer agents (such as the well-known anticancer agents docetaxel, cisplatin, and/or gemcitabine) have shown good anticancer activity.

The present inventors have shown that anti-AMHRII antibodies having antitumor efficacy against AMHRII expressing gynaecological cancers have also been demonstrated in the art to be useful in the prevention or treatment of AMHRII expressing lung cancer, and especially those in the disclosure herein that express AMHRII, such as non-small cell lung cancer, especially epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC, polymorphous cell carcinoma NSCLC and neuroendocrine NSCLC.

More precisely, in the examples herein, it is shown that the anti-AMHRII antibody, named 3C23K, exerts anti-tumor activity in vivo on human lung cancer, especially on non-small cell lung cancer as disclosed herein, including when the anti-AMHRII antibody treatment is combined with treatment with one or more different anti-cancer agents, such as docetaxel, cisplatin, and/or gemcitabine.

Still further, the inventors have shown that anti-AMHRII C23K antibodies do not induce detectable toxic events in vivo, particularly do not induce significant weight loss.

The present invention therefore relates to the use of a human AMHRII binding agent for the prevention or treatment of lung cancer, in particular non-small cell lung cancer (NSCLC), more in particular non-small cell lung cancer (NSCLC) selected from the group comprising: epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCLC, polymorphous cell carcinoma and neuroendocrine NSCLC.

The invention also relates to the use of a human AMHRII binding agent for the manufacture of a medicament for the prevention or treatment of lung cancer, in particular lung cancer selected from the group comprising: epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCLC, polymorphic cell carcinoma NSCLC and neuroendocrine NSCLC.

The invention also relates to a method for preventing or treating lung cancer, in particular lung cancer selected from the group comprising: an epidermoid NSCLC, an adenocarcinoma NSCLC, a large cell NSCLC, a squamous cell carcinoma NSCLC, a polymorphous cell carcinoma NSCLC, and a neuroendocrine NSCLC, wherein the method comprises the step of administering a AMHRII binding agent disclosed in the present specification to a subject in need thereof.

AMHRII binders that can be used according to the present invention do not need to mimic MIS natural ligand activity. Thus, AMHRII binders that can be used according to the present invention do not require activation of any cell signaling pathway after binding to AMHRII. In contrast, only the agent is required to have the ability to bind AMHRII, as the agent is specifically used to target cytotoxicity inducing activity (such as a cytotoxicity inducing entity) that encompasses anti-AMHRII cytotoxic immunoconjugates, ADCC-induced anti-AMHRII antibodies or ADC-induced anti-AMHRII antibodies or CAR T cells expressing AMHRII binding engineered receptors.

AMHRII binding agents

As used herein, AMHRII binding agents encompass any such agent that specifically binds to AMHRII and when presented in an appropriate manner will cause death of target cells that express AMHRII at their surface after the agent binds to AMHRII expressed by the cell membrane.

AMHRII binders as described herein for use in treating lung cancer may also be referred to herein as "therapeutic AMHRII binders".

Typically, AMHRII binders encompass proteins or nucleic acids that specifically bind to AMHRII.

AMHRII binding proteins predominantly encompass proteins comprising one or more Complementarity Determining Regions (CDRs) derived from the AMHRII binding fragment of an anti-AMHRII antibody or an anti-AMHRII antibody, it being understood that the AMHRII binding proteins can be expressed as Chimeric Antigen Receptors (CARs) by engineered cells such as CAR-T cells, NK T cells, or CAR macrophages.

AMHRII binding nucleic acids encompass predominantly nucleic acid aptamers specifically selected for their specific binding properties to AMHRII.

In some preferred embodiments, the AMHRII binding agent is an anti-AMHRII antibody or AMHRII binding fragment thereof.

In a most preferred embodiment, the AMHRII binding agent is an anti-AMHRII monoclonal antibody or AMHRII binding fragment thereof.

According to these preferred embodiments, anti-AMHRII monoclonal antibodies encompass chimeric anti-AMHRII antibodies, humanized anti-AMHRII antibodies and human AMHRII antibodies, as well as AMHRII-binding fragments and AMHRII-binding derivatives thereof.

Various AMHRII antibodies are known in the art and may be used as AMHRII binding agents according to the invention. For the purposes of practicing the present invention, one skilled in the art can use recombinant human anti AMHRII, sold by Creative Biolabs under the number No. mhh-57 for purposes of illustration.

In some embodiments, the anti-AMHRII antibody that can be used according to the invention is a humanized 12G4 antibody disclosed in PCT application No. wo 2008/053330.

In some other embodiments, the anti-AMHRII antibody is a humanized antibody described in PCT application No. wo 2011/141653, which encompasses 3C23 antibodies, as well as variants thereof, including 3C23K humanized antibodies.

In a still further embodiment, the anti-AMHRII antibodies are those described in PCT application No. wo 2017/025458. According to these further embodiments, PCT application No. wo 2017/025458 discloses AMHRII binders in the form of Antibody Drug Conjugates (ADCs), wherein the anti-AMHRII antibodies are linked to a cytotoxic agent.

Monoclonal antibodies directed against the muller hormone type II receptor (and humanized derivatives thereof) have been developed in the art for the treatment of ovarian cancer (see EP 2097453B1 and U.S. patent No.8,278,423, which are incorporated herein by reference in their entirety).

Among AMHRII binders that can be used according to the invention, monoclonal antibody 12G4 (mAb 12G 4) or chimeric or humanized variants thereof, including antibodies derived with drugs or detectable labels to form ADCs, can be used by those skilled in the art. The mAbl2G4 producing hybridoma was deposited with the national center for microbiological bacterial collection (CNCM, pasteur institute, france 75724, area 15, kluyvere street 25) under the terms of the Budapest treaty at month 9 and 26 of 2006, and the CNCM deposit number was 1-3673. The variable domains of the light and heavy chains of mAb 12G4 have been sequenced to be the Complementarity Determining Regions (CDRs) of mAb 12G4 (see EP 2097453B1 and U.S. patent No.8,278,423, which are incorporated herein by reference in their entirety). mAb 12G4 and chimeric or humanized variants thereof can be used to produce an ADC as disclosed herein.

PCT application No. PCT/FR2011/050745 (international publication No. wo/2011/141653) and U.S. patent No.9,012,607 (each of which is incorporated herein by reference in its entirety) disclose novel humanized antibodies derived from murine 12G4 antibodies. For the purposes of the present invention, these humanized antibodies may be used as AMHRII binders. In a specific embodiment disclosed in PCT application No. wo/2011/141653, antibodies are those identified as 3C23 and 3C 23K. The nucleic acid sequences and polypeptide sequences of these antibodies are provided herein as SEQ ID NO. 1 through SEQ ID NO. 16. In some aspects of the invention, an anti-AMHRII antibody of interest may be referred to as "comprising a light chain comprising SEQ ID NO: and a heavy chain comprising SEQ ID NO:". Thus, in various embodiments, particularly preferred antibodies (including antibodies for use in the production of ADCs) include:

a) A light chain comprising SEQ ID NO. 2 and a heavy chain comprising SEQ ID NO.4 (3C 23 VL sequence and 3C23 VH sequence without leader sequence);

b) A light chain comprising SEQ ID NO. 6 and a heavy chain comprising SEQ ID NO. 8 (3C 23K VL sequence and 3C23K VH sequence without leader sequence);

c) A light chain comprising SEQ ID NO. 10 and a heavy chain comprising SEQ ID NO. 12 (3C 23 light chain and 3C23 heavy chain without leader sequence);

d) A light chain comprising SEQ ID NO. 14 and a heavy chain comprising SEQ ID NO. 16 (3C 23K light chain and 3C23K heavy chain without a leader sequence).

Other antibodies (e.g., humanized or chimeric) may be used as anti-MAHRII binding agents of interest, including antibodies for forming ADCs, based on the heavy and light chain sequences provided in fig. 1A and 1B (e.g., antibodies, such as humanized or chimeric antibodies comprising the CDR sequences disclosed in the figures). Thus, the invention also relates to the use of an anti-AMHRII antibody comprising/consisting of CDRs comprising (or consisting of) the following sequences:

CDRL-1: RASX1X2VX3X4X5A (SEQ ID No. 65), wherein X1 and X2 are independently S or P, X3 is R or W or G, X4 is T or D, and X5 is I or T;

-CDRL-2 is PTSSLX S (SEQ ID No. 66), wherein X6 is K or E; and

-CDRL-3 is LQWSSYPWT (SEQ ID No. 67);

-CDRH-1 is KASGYX FTX8X9HIH (SEQ ID No. 68), wherein X7 is S or T, X is S or G and X9 is Y or N;

-CDRH-2 is WIYPX10DDSTKYSQKFQG (SEQ ID No. 69), wherein X10 is G or E; and

CDRH-3 is GDRFAY (SEQ ID NO. 70).

The invention also relates to the use of ADCs produced using such anti-AMHRII antibodies for the treatment of lung cancer, particularly non-small cell lung cancer and small cell lung cancer.

Antibodies (e.g., chimeric or humanized antibodies) within the scope of the application include those disclosed in the following table: alternatively, human monoclonal antibodies that specifically bind to AMHR-II may be used to make ADCs. The 3C23K antibody is defined by:

SEQ ID NO. 19 for the VH amino acid sequence,

SEQ ID NO. 36 for the VL amino acid sequence.

Table 1 below lists anti-AMHRII humanized antibodies that can be used according to the invention.

Table 1: anti-AMHRII antibodies

Anti-AMHRII antibodies, AMHRII binding fragments or AMHRII binding derivatives of anti-AMHRII antibodies

The term "antibody" is used in its broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (see below) so long as they exhibit the desired biological activity.

Thus, as used herein, the term "antibody" refers collectively to an immunoglobulin or immunoglobulin-like molecule, including by way of example and without limitation IgA, igD, igE, igG, and IgM, combinations thereof, and similar molecules (such as shark immunoglobulins) produced during an immune response in any vertebrate (e.g., mammals such as humans, goats, rabbits, and mice, as well as non-mammalian species). Unless specifically stated otherwise, the term "antibody" includes intact immunoglobulins and "antibody fragments" or "antigen-binding fragments" that specifically bind to AMHRII, essentially excluding binding to other molecules (i.e., molecules not related to AMHRII). The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies). See also Pierce Catalog and Handbook,1994-1995 (PIERCE CHEMICAL co., rockford, 111.); kuby, j., immunology, 7 th edition, w.h. freeman & co., new york, 2013.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single antigen. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring antibody production by any particular method. For example, monoclonal antibodies to be used according to the present invention may be prepared by the hybridoma method described first by Kohler et al, nature 256:495 (1975), or may be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described by Clackson et al, nature 352:624-628 (1991) or Marks et al, J.MoI biol.222:581-597 (1991).

The term "antibody fragment" refers to a portion of an intact antibody and to the epitope variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, fab fragments, fab 'fragments, F (ab') 2 fragments, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation.

As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, with kappa light chain and lambda light chain referring to the two major antibody light chain isotypes.

As used herein, the term "complementarity determining region" or "CDR" refers to the portion of two variable chains (heavy and light chains) of an antibody that recognize and bind to a particular antigen. CDRs are the most variable part of the variable chain and provide the antibody with its specificity. Three CDRs are on each of the Variable Heavy (VH) and Variable Light (VL) chains, thus giving a total of six CDRs per antibody molecule. CDRs are mainly responsible for binding to epitopes of antigens. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, VHCDR3 is located in the variable domain of the antibody heavy chain for which it is found, while VLCDR1 is CDR1 from the variable domain of the antibody light chain for which it is found. Antibodies that bind LHR will have specific VH and VL region sequences and therefore specific CDR sequences. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions in the CDRs are directly involved in antigen binding. These positions in the CDRs are called Specificity Determining Residues (SDRs).

"Framework regions" (hereinafter referred to as FRs) are those variable domain residues that differ from the CDR residues. Each variable domain typically has four FR identified as FR1, FR2, FR3 and FR 4. If the CDRs are defined according to Kabat, the light chain FR residues are located at about residues 1-23 (LCFR 1), 35-49 (LCFR 2), 57-88 (LCFR 3) and 98-107 (LCFR 4), and the heavy chain FR residues are located at about residues 1-30 (HCFR 1), 36-49 (HCFR 2), 66-94 (HCFR 3) and 103-113 (HCFR 4) of the heavy chain residues.

"Single chain Fv" or "scFv" antibody fragments include the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, fv polypeptides also include a polypeptide linker between the VH domain and the VL domain, which allows the scFv to form the desired antigen binding structure. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, volume 113, rosenburg and Moore, springer-Verlag, new York, pages 269-315 (1994).

The term "diabody" refers to a small antibody fragment having two antigen-binding sites, said fragment comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH and VL). By using a linker that is too short to pair between two domains on the same strand, the domains are forced to pair with the complementary domain of the other strand and form two antigen binding sites. Diabodies are described in, for example, EP 404,097; WO 93/11161; and Hollinger et al, proc.Natl. Acad. Sci. USA,90:6444-6448 (1993).

Diabodies or bispecific antibodies can be broadly divided into two classes: immunoglobulin G (IgG) -like molecules and non-IgG-like molecules. IgG-like bsAb retains Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell phagocytosis (ADCP) (Spiess et al, 2015, mol Immunol.,. 67 (2) vol.: 95-106.). The Fc region of bsAb aids in purification and improves solubility and stability. Bispecific antibodies in IgG-like format generally have longer serum half-lives due to their larger size and FcRn-mediated recycling (Kontermann et al, 2015,Bispecific antibodies.Drug Discov Today, volume 20 (7): 838-47). The smaller size of the non-IgG-like bsAb results in enhanced tissue penetration (Kontermann et al, 2015,Bispecific antibodies.Drug Discov Today, volume 20 (7): 838-47).

According to some preferred embodiments, the bispecific antibody according to the invention comprises: (i) A first antigen binding site that binds to AMHRII, and (ii) a second antigen binding site that binds to a target antigen other than AMHRII, particularly a target antigen that can be expressed by cancer cells or immune cells of the tumor environment (such as T cells, NK cells, or macrophages). In some embodiments, in such bispecific antibodies, the second antigen binding site binds to a target antigen (which is CD 3) and binds T cells. The target antigen may also be PDL1 to unlock T cells, or CD16 to activate NK or macrophages.

Monoclonal antibodies specified herein include, inter alia, "chimeric" anti-AMHRII antibodies (immunoglobulins) in which a portion of the heavy and/or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; and Morrison et al, proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

The monoclonal antibodies specified herein also encompass humanized anti-AMHRII antibodies. A "humanized" version of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequences derived from a non-human immunoglobulin. In most cases, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced with residues from the hypervariable region of a non-human species (donor antibody, such as mouse, rat, rabbit or non-human primate) having the desired specificity, affinity, and capability. In some cases, fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or the donor antibody. These modifications were made to further refine antibody performance. Typically, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional details, please see Jones et al, nature 321:522-525 (1986); riechmann et al Nature 332:323-329 (1988); and Presta, curr.Op.struct.biol.2:593-596 (1992).

Monoclonal anti-AMHRII antibodies specified herein also encompass anti-AMHRII human antibodies. A "human antibody" is a human antibody having an amino acid sequence corresponding to that of an antibody produced by a human and/or made using any of the techniques for making a human antibody as disclosed herein. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues. Human antibodies can be produced using a variety of techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses human antibodies (Vaughan et al Nature Biotechnology 14:309-314 (1996): sheets et al Proc. Natl. Acad. Sci.95:6157-6162 (1998)); hoogenboom and Winter, J.MoI.biol,227:381 (1991); marks et al, J.MoI.biol,222:581 (1991)). Human antibodies can also be made by introducing a human immunoglobulin locus into a transgenic animal (e.g., a mouse) in which endogenous immunoglobulin genes have been partially or fully inactivated. Upon challenge, human antibody production was observed, which was very similar in all respects to that seen in humans, including gene rearrangement, assembly, and antibody profiling. This method is described, for example, in U.S. patent No.5,545,807;5,545,806;5,569,825;5,625,126;5,633,425;5,661,016 and the following scientific publications: marks et al, bio/Technology 10:779-783 (1992); lonberg et al, nature 368:856-859 (1994); morrison, nature 368:812-13 (1994); fishwild et al Nature Biotechnology 14:845-51 (1996); neuberger, nature Biotechnology 14:826 (1996); lonberg and Huszar, international Rev. Immunol.13:65-93 (1995). Alternatively, human antibodies may be prepared via immortalization of human B lymphocytes that produce antibodies to the target antigen (such B lymphocytes may be recovered from the individual or may be immunized in vitro). See, e.g., cole et al Monoclonal Antibodies AND CANCER THERAPY, alan R.Lists, page 77 (1985); boerner et al, J.Immunol,147 (l): 86-95 (1991); and U.S. patent No.5,750,373.

As used herein, "antibody mutant" or "antibody variant" refers to an amino acid sequence variant of a species-dependent antibody in which one or more of the amino acid residues of the species-dependent antibody have been modified. Such mutants must have less than 100% sequence identity or similarity to the species-dependent antibody. In one embodiment, the antibody mutant will have an amino acid sequence that has at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90% and most preferably at least 95% amino acid sequence identity or similarity to the amino acid sequence of the heavy chain variable domain or the light chain variable domain of a species-dependent antibody. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in a candidate sequence that are identical (i.e., identical residues) or similar (i.e., amino acid residues from the same group, based on common side chain properties, see below) to the species-dependent antibody residue after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity. Antibody sequences outside of the N-terminal, C-terminal or internal extension, deletion or insertion variable domains should not be construed as affecting sequence identity or similarity.

Humanized antibodies can be produced by obtaining nucleic acid sequences encoding CDR domains and constructing humanized antibodies according to techniques known in the art. Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, e.g., riechmann l. Et al 1988;Neuberger M S, et al, 1985). Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. No.5,225,539;5,530,101; and 5,585,089), veneering (veneering) or surface remodeling (resurfacing) (EP 592,106;EP 519,596;Padlan E A (1991); studnicka G M et al (1994); roguska M A et al (1994)) and chain replacement (shuffling) (U.S. Pat. No.5,565,332). Universal recombinant DNA techniques for the preparation of such antibodies are also known (see european patent application EP 125023 and international patent application WO 96/02576).

It may be desirable to modify an anti-AMHRII antibody specified herein with respect to effector function, for example, to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be accomplished by introducing one or more amino acid substituents into the Fc region of the antibody. Alternatively or additionally, cysteine residues may be introduced in the Fc region, thereby allowing inter-chain disulfide bond formation within that region. Homodimeric antibodies thus produced may have improved internalization ability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J.exp Med.176:1191-1195 (1992) and Shopes, B.J.Immunol.148:2918-2922 (1992). Heterodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linking reagents, as described in Wolff et al CANCER RESEARCH 53:2560-2565 (1993). Alternatively, the antibody may be engineered with a double Fc region and thus may have enhanced complement lysis and ADCC capabilities. See Stevenson et al, anti-Cancer Drug Design, 3:219-230 (1989). WO00/42072 (Presta, l.) describes antibodies with improved ADCC function in the presence of human effector cells, wherein the antibodies comprise amino acid substitutions in their Fc region. Preferably, the antibody with improved ADCC comprises substitutions at positions 298, 333 and/or 334 (Eu numbering of residues) of the Fc region. Preferably, the altered Fc region is a human IgG1 Fc region comprising or consisting of substitutions at one, two or three of these positions. Such substitutions are optionally combined with substitutions that increase CIq binding and/or CDC.

Antibodies with altered CIq binding and/or Complement Dependent Cytotoxicity (CDC) are described in WO99/51642, U.S. Pat. No.6,194,551Bl, U.S. Pat. No.6,242,195Bl, U.S. Pat. No.6,528,624Bl and U.S. Pat. No.6,538,124 (Idusogie et al). The antibody comprises amino acid substitutions at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333 and/or 334 (Eu numbering of residues) of its Fc region.

In some embodiments, AMHRII binders encompass glycoengineered anti-AMHRII antibodies.

As used herein, the term "glycoengineering" refers to any art-recognized method for altering the glycoform profile (glycoform profile) of a binding protein composition. Such methods include expressing the binding protein composition in a genetically engineered host cell (e.g., CHO cell) genetically engineered to express a heterologous glycosyltransferase or glycosidase. In other embodiments, the methods of glycoengineering comprise culturing the host cell under conditions biased towards a particular glycoform profile.

As used herein, "glycoengineered antibody" encompasses (i) antibodies comprising a hypergalactosylated Fc fragment, (ii) antibodies comprising a low mannosylated Fc fragment (which encompasses a mannosylated Fc fragment), and (iii) antibodies comprising a low fucosylated Fc fragment (which encompasses a fucosylated Fc fragment). As used herein, a glycoengineered fragment encompasses an Fc fragment with altered glycosylation selected from the group comprising one or more of the following altered glycosylation: (i) hypergalactosylation, (ii) low mannosylation and (iii) low fucosylation. As a result, the glycoengineered Fc fragment from an anti-AMHRII antibody as used in accordance with the invention encompasses illustrative examples of high galactosylated Fc fragments, low mannosylated Fc fragments, and low fucosylated Fc fragments.

One skilled in the art can refer to the known techniques for obtaining anti-AMHRII antibodies comprising a highly galactosylated Fc fragment, a low mannosylated Fc fragment, and a low fucosylated Fc fragment that are known to bind to Fc receptors with higher affinity than the unmodified Fc fragment.

The glycoengineered anti-AMHRII antibodies encompass anti-AMHRII antibodies comprising a low fucosylated Fc fragment (which may also be referred to as a "low fucose" Fc fragment).

Immunoconjugates, in particular Antibody Drug Conjugates (ADCs)

AMHRII binders that can be used for the purposes of the present invention encompass antibodies specified herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioisotope (i.e., a radioactive conjugate). Such antibody conjugates encompass those described in PCT application No. wo 2017/025458. PCT application No. wo 2017/025458 discloses, inter alia, anti-AMHRII C23K antibodies, as well as 3C23K ADC conjugates, which are shown herein to have in vivo anti-cancer activity against non-gynaecological human cancers.

Cytotoxic agents encompass enzymatically active toxins. Enzymatically active toxins and fragments thereof that may be used include diphtheria chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from pseudomonas aeruginosa (Pseudomonas aeruginosa)), ricin A chain, abrin A chain, mo Disu A chain, α -sarcina, jatropha curcas (aleurous fordii) protein, carnation, pokeweed protein (PAPI, PAPII and PAP-S), balsam pear (momordica charantia) inhibitors, curcin, crotonin, soapbox (sapaonaria officinalis) inhibitors, gelonin, mi Tuojun, curcin, phenol, enomycin and trichothecene.

A variety of radionuclides can be used to produce the radioconjugate antibodies.

Conjugates of antibodies and cytotoxic agents are prepared using a variety of bifunctional protein coupling agents such as those disclosed in PCT application No. wo 2017/025458.

Preferred immunoconjugates of the anti-AMHRII ADC antibody conjugates are those described in PCT application No. wo 2017/025458.

CAR cells, including CAR T cells, CAR NK cells, and CAR macrophages

In some embodiments, the human AMHRII binding agent is a AMHRII-binding receptor or AMHRII-binding receptor-expressing cell, particularly a AMHRII-binding receptor-expressing CAR T cell, a AMHRII-binding receptor NK cell, or a AMHRII-binding receptor-expressing CAR macrophage.

Thus, in some embodiments, the human AMHRII binding agent is AMHRII binding to an engineered receptor, most preferably AMHRII binding domain thereof AMHRII binding domain derived from a monoclonal anti-AMHRII antibody disclosed in this specification binds to an engineered receptor.

Typically, AMHRII binding engineered receptors consist of Chimeric Antigen Receptors (CARs) comprising: (i) an extracellular domain, (ii) a transmembrane domain, and (iii) an intracellular domain, and wherein the extracellular domain is a AMHRII binding moiety derived from an anti-AMHRII monoclonal antibody disclosed in the present specification. In some embodiments, the AMHRII-binding extracellular domain of an engineered receptor comprises (i) an antibody VH chain comprising CDRs derived from an anti-AMHRII monoclonal antibody disclosed herein and (ii) an antibody VL chain comprising CDRs derived from an anti-AMHRII monoclonal antibody disclosed herein. In some embodiments, the AMHRII-binding extracellular domain of an engineered receptor comprises a VH chain and a VL chain of an anti-AMHRII monoclonal antibody disclosed herein. In some embodiments, the AMHRII-binding extracellular domain of an engineered receptor is an ScFv comprising CDRs derived from a VH chain and a CH chain, respectively, from an anti-AMHRII monoclonal antibody disclosed in the present specification. In some embodiments, the AMHRII extracellular domain that binds to an engineered receptor is an ScFv comprising a VH chain and a CH chain from an anti-AMHRII monoclonal antibody disclosed in the present specification, respectively.

Also encompassed herein are AMHRII binding agents comprised of cells expressing such AMHRII-binding receptors, particularly CAR T cells, CAR NK cells, or CAR macrophages expressing such AMHRII-binding receptors.

The term "chimeric antigen receptor" (CAR) as used herein refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide from which the extracellular domain is derived, and at least one intracellular domain. "Chimeric Antigen Receptor (CAR)" is sometimes also referred to as "chimeric receptor", "T-body" or "Chimeric Immune Receptor (CIR)". By "capable of binding to the extracellular domain of AMHRII" is meant any oligopeptide or polypeptide that can bind to AMHRII. By "intracellular domain" is meant any oligopeptide or polypeptide known in cells to be used as a domain for transmitting signals to cause activation or inhibition of biological processes. By "transmembrane domain" is meant any oligopeptide or polypeptide which is known to span the cell membrane and which can be used to connect an extracellular domain to a signaling domain. Chimeric antigen receptors may optionally comprise a "hinge domain" that serves as a linker between the extracellular domain and the transmembrane domain.

CAR T cells are genetically engineered autologous T cells in which a single chain antibody fragment (scFv) or ligand is attached to a T cell signaling domain capable of promoting T cell activation (Maher, J. (2012) ISRN Oncol.2012:278093; curran, K.J. et al (2012) J.Gene Med.14:405-415; fedorov, V.D. et al (2014) Cancer J.20:160-165; barrett, D.M. et al (2014) Annu. Rev. Med.65:333-347).

By "intracellular signaling domain" is meant the portion of the CAR that is found or engineered to be found within a T cell. The "intracellular signaling domain" may or may not further comprise a "transmembrane domain" that anchors the CAR in the plasma membrane of a T cell. In one embodiment, the "transmembrane domain" and the "intracellular signaling domain" are derived from the same protein (e.g., cd3ζ); in other embodiments, the intracellular signaling domain and the transmembrane domain are derived from different proteins (e.g., the transmembrane domain of cd3ζ and the intracellular signaling domain of CD28 molecule, and vice versa).

"Costimulatory domain (endodomain)" means the intracellular signaling domain derived from a T cell costimulatory molecule or a fragment thereof. A non-limiting list of T cell costimulatory molecules includes CD3, CD28, OX-40, 4-1BB, CD27, CD270, CD30, and ICOS. The co-stimulatory domains may or may not include transmembrane domains from the same or different co-stimulatory domains.

By "extracellular antigen binding domain" is meant that portion of the CAR that specifically recognizes AMHRII and binds to AMHRII.

In a preferred embodiment, the "extracellular binding domain" is derived from an anti-AMHRII monoclonal antibody. For example, an "extracellular binding domain" may include all or part of a Fab domain from a monoclonal antibody. In certain embodiments, an "extracellular binding domain" includes a complementarity determining region of a particular anti-AMHRII monoclonal antibody. In yet another embodiment, the "extracellular binding domain" is a single chain variable fragment (scFv) obtained from an anti-AMHRII monoclonal antibody specified herein.

In a preferred embodiment, the extracellular binding domain is derived from any of the anti-AMHRII monoclonal antibodies described in the present specification, in particular from the 3C23K anti-AMHRII monoclonal antibody.

I. Extracellular antigen binding domains

In one embodiment, the CAR of the invention comprises an extracellular antigen-binding domain from one of the anti-AMHRII monoclonal antibodies described herein.

In one embodiment, the extracellular binding domain comprises the following CDR sequences:

CDRL-1: RASX1X2VX3X4X5A (SEQ ID NO. 65), wherein X1 and X2 are independently S or P, X3 is R or W or G, X4 is T or D, and X5 is I or T;

-CDRL-2 is PTSSLX S (SEQ ID No. 66), wherein X6 is K or E; and

-CDRL-3 is LQWSSYPWT (SEQ ID No. 67);

-CDRH-1 is KASGYX FTX8X9HIH (SEQ ID No. 68), wherein X7 is S or T, X is S or G and X9 is Y or N;

CDRH-2 is WIYPX10DDSTKYSQKFQG (SEQ ID NO. 69), wherein X10 is G or E, and

CDRH-3 is GDRFAY (SEQ ID NO. 70).

Linker between VL and VH domains of kappa Mab scFv

In another embodiment, the antibody AMHRII VL is attached to the antibody AMHRII VH via a flexible linker. In particular, the flexible linker is a glycine/serine linker of about 10 to about 30 amino acids (e.g., 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids) and comprises the structure (Gly 4 Ser) ³.

III spacer between extracellular antigen binding Domain and intracellular Signal transduction Domain

The extracellular antigen binding domain is linked to the intracellular signaling domain through the use of a "spacer". The spacer is designed to be flexible enough to allow the antigen binding domain to be oriented in a manner that aids in antigen recognition and binding. The spacer may be derived from the anti-AMHRII immunoglobulin itself and may include an IgGl hinge region or the CH2 and/or CH3 regions of IgG.

Intracellular signaling domains

The intracellular signaling domain comprises all or part of the CD3 chain. CD is also known as CD247, together with CD 4T cell co-receptors or CD 8T cell co-receptors, is responsible for coupling extracellular antigen recognition to the intracellular signaling cascade.

In addition to including the CD3 zeta signaling domain, inclusion of a costimulatory molecule has been shown to enhance CAR T cell activity in murine models and clinical trials. Several have been studied, including CD28, 4-IBB, ICOS, CD, CD270, CD30, and OX-40.

In certain embodiments, a method of producing a CAR-expressing cell is disclosed, the method comprising, or alternatively consisting essentially of: (i) Transducing the isolated population of cells with a nucleic acid sequence encoding a CAR, and (ii) selecting a subpopulation of cells that have been successfully transduced with the nucleic acid sequence of step (i). In some embodiments, the isolated cell is a T cell, an animal T cell, a mammalian T cell, a feline T cell, a canine T cell, or a human T cell, thereby producing a CAR T cell. In certain embodiments, the isolated cell is an NK cell, e.g., an animal NK cell, a mammalian NK cell, a feline NK cell, a canine NK cell, or a human NK cell, thereby producing a CAR NK cell.

Therapeutic uses of CAR T cells, CAR NK T cells, and CAR macrophages

CAR cells including CAR T cells, CAR NK cells, and CAR macrophages described herein can be used to treat AMHRII-expressing lung tumors. The CAR cells of the invention are preferably used to treat AMHRII-expressing lung tumors in patients with lung cancer, particularly non-small cell lung cancer or small cell lung cancer, as described herein.

The CAR cells of the invention can be administered alone or in combination with diluents, known anti-cancer therapeutic agents and/or other components (such as cytokines) or other cell populations having immunostimulation.

The method aspects of the invention relate to methods for inhibiting tumor growth in a subject in need thereof and/or for treating a cancer patient in need thereof. In some embodiments, the tumor is a solid lung tumor.

The CAR cells as disclosed herein can be administered alone or in combination with diluents, known anti-cancer therapeutic agents and/or other components (such as cytokines) or other cell populations that are immunostimulatory. They may be first line therapy, second line therapy, third line therapy, fourth line therapy or another therapy. They may be combined with other therapies. Non-limiting examples of such include chemotherapy or biological agents. The appropriate treatment regimen will be determined by the attending physician or veterinarian.

The pharmaceutical composition comprising the CAR of the invention may be administered in a manner suitable for the disease to be treated or prevented. Although the appropriate dosage may be determined by clinical trials, the amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease.

Therapeutic applications

As already disclosed elsewhere in this specification, the AMHRII binding agent disclosed herein encompasses (i) an anti-AMHRII antibody disclosed herein, (ii) an antibody drug conjugate disclosed herein and (iii) a CAR cell disclosed herein (including CAR T cells, CAR NK cells and CAR macrophages), the AMHRII binding agent consisting of an active ingredient that can be used to prevent or treat AMHRII-expressing lung cancer, particularly non-small cell lung cancer (NSCLC), more precisely NSCLC selected from the group comprising: epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC, and neuroendocrine NSCLC.

Methods of cancer treatment using anti-tumor antigen antibodies or anti-tumor antigen CAR cells are well known to those skilled in the art.

In some embodiments, cancer patients are tested to determine if their tumor cells express AMHRII at their surface prior to treatment with AMHRII binding agents (such as anti-AMHRII antibodies, anti-AMHRII ADC or anti-AMHRII CAR T cells).

Such preliminary tests for detecting AMHRII membrane expression are preferably used to treat lung cancer with low frequency expression AMHRII. In contrast, for the treatment of cancers with high frequency expression AMHRII (such as the illustrated epidermoid NSCLC), such preliminary tests for detecting membrane expression of AMHRII may not be performed.

Thus, in some embodiments, the invention relates to the use of AMHRII binding agents as specified herein for the prevention or treatment of an individual having AMHRII-positive lung cancer, including non-small cell lung cancer (NSCLC), in particular NSCLC selected from the group comprising: epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCLC, polymorphic cell carcinoma NSCLC and neuroendocrine NSCLC.

The present invention relates to the use of AMHRII binding agents for the manufacture of a medicament for the prevention or treatment of a subject suffering from AMHRII-positive lung cancer, including non-small cell lung cancer (NSCLC), in particular NSCLC selected from the group comprising: epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCL, polymorphic cell carcinoma NSCLC C and neuroendocrine NSCLC.

The invention also relates to a method for preventing or treating an individual having AMHRII-positive lung cancer, including non-small cell lung cancer (NSCLC), in particular NSCLC selected from the group comprising: epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC, squamous cell carcinoma NSCLC, polymorphic cell carcinoma NSCLC, and neuroendocrine NSCLC, wherein the method comprises the step of administering an anti-AMHRII binding agent to the individual.

An individual may be designated as having AMHRII-positive cancer by performing a method of detecting cell surface AMHRII protein expression on a lung cancer tissue sample previously obtained from the individual. Detection of cell surface AMHRII protein expression can be performed according to a variety of methods well known to those skilled in the art. Cell surface AMHRII protein expression detection methods specifically encompass immunohistochemical methods as well as fluorescence activated cell sorting methods, as shown in the examples herein.

The invention also relates to a method for determining whether an individual is suitable for lung cancer treatment with AMHRII binding agent (i.e., whether an individual is responsive to lung cancer treatment with AMHRII binding agent), wherein the method comprises the step of determining whether a lung tumor tissue sample previously obtained from the individual expresses AMHRII protein at the cell surface.

Accordingly, the present invention also relates to a method for determining whether an individual suffering from lung cancer, in particular non-small cell lung cancer (NSCLC), in particular NSCLC selected from the group comprising epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC and neuroendocrine NSCLC is suitable for cancer treatment with a AMHRII binding agent (i.e. in response to cancer treatment with a AMHRII binding agent), wherein the method comprises the steps of:

a) Determining whether cancer cells from said patient are expressed at their membrane AMHRII, and

B) And (3) deducing: if the membrane expression of AMHRII of the lung cancer cells is determined in step a), the patient is suitable for lung cancer treatment with the AMHRII binding agent (i.e., is responsive to lung cancer treatment with the AMHRII binding agent).

In a preferred embodiment of the method, in step b), the patient is inferred to be suitable (i.e., responsive) to treatment of lung cancer with a AMHRII binding agent when (i) the AMHRII expression score value is determined in step a) and when (ii) the AMHRII expression score value is a threshold score value or higher. The AMHRII score is most preferably calculated using formula (I) as described elsewhere in this specification.

Thus, according to a preferred embodiment, step a) of the method is performed by an immunohistochemical method, such as shown in the examples herein.

The cancer cells used in step a) are typically derived from a biopsy tissue sample previously collected from the cancer patient.

Preferably, step a) is performed by using an anti-AMHRII antibody, in particular a 3C23K antibody, selected from those specifically described in the present specification, the AMHRII binding of which can be detected by using a labeled secondary antibody according to well known antibody detection techniques, such as those disclosed in the examples herein.

Preferably, when a scoring method that allows determination of the E score value is performed according to the following formula (I), when it is determined that the membrane AMHRII expression score value is 1.0 or greater in a cancer cell sample derived from a cancer patient having lung cancer included in the lung cancer group listed above, the patient is determined to be suitable (i.e., responsive) to treatment of lung cancer with a AMHRII binding agent:

e score = frequency x AMHRII _level, where

The E score means a membrane AMHRII expression score value for a given cancer cell sample,

Frequency means the frequency of the cells contained in the lung cancer cell sample in which membrane AMHRII expression is detected, and

-AMHRII _level means the level of AMHRII membrane expression of AMHRII expressing cells contained in the given lung cancer cell sample.

Accordingly, the present invention also relates to a method for treating a patient suffering from non-small cell lung cancer (NSCLC), wherein the method comprises the steps of:

a) Determining whether a tumor tissue sample previously obtained from said individual expresses AMHRII protein at the cell surface, and

B) If cell surface expression of AMHRII has been determined in step a), the individual is treated with AMHRII binding agent.

In a most preferred embodiment, in step a), AMHRII expression is determined when the tumor sample has a membrane AMHRII expression score value "E score" (covering an E score value of 1.5 or greater) calculated according to formula (I) above.

In a most preferred embodiment of the above method, the AMHRII binding agent consists of an anti-AMHRII antibody or fragment thereof as specified herein, or consists of a CAR cell (e.g., CAR T cell or CAR NK cell) as specified herein.

In some embodiments, the AMHRII binding agent is used as the sole anticancer active ingredient.

In some other embodiments, the anti-cancer treatment with the AMHRII binding agent further comprises subjecting the individual to one or more additional anti-cancer treatments, including radiation therapy treatment and chemotherapeutic agent treatment.

Thus, according to such other embodiments, the anti-cancer treatment with the AMHRII binding agent further comprises administering to the individual one or more additional anti-cancer active ingredients.

Combination therapy

As shown in the examples herein, effective anti-lung cancer lung therapies encompass those in which an anti-AMHRII monoclonal antibody is combined with one or more different anti-cancer agents. The examples herein illustrate combination therapies for lung cancer wherein an anti-AMHRII antibody is combined with docetaxel or with a combination of cisplatin and gemcitabine.

An "anticancer agent" is defined as any molecule that can interfere with the biosynthesis of macromolecules (DNA, RNA, proteins, etc.) or inhibit cell proliferation or cause cell death (e.g., by apoptosis or cytotoxicity). Among the anticancer agents that may be mentioned are alkylating agents, topoisomerase inhibitors and intercalators, antimetabolites, lysing agents, agents interfering with tubulin, monoclonal antibodies.

By "pharmaceutically acceptable vehicle" is meant a non-toxic substance that is compatible with a biological system, such as a cell, cell culture, tissue or organism.

According to a particular aspect, the present invention relates to a pharmaceutical composition comprising an anticancer agent as active ingredient in combination with a pharmaceutically acceptable vehicle and an antibody binding to AMHR-II, in particular an anti-AMHRII antibody as described herein.

In some embodiments, the invention relates to pharmaceutical compositions comprising an anticancer agent as an active ingredient in combination with a pharmaceutically acceptable vehicle and an antibody that binds AMHR-II, in particular an anti-AMHRII antibody described herein.

In some embodiments, the invention relates to a pharmaceutical composition comprising an anticancer agent and an antibody that binds AMHR-II as active ingredients in combination with a pharmaceutically acceptable vehicle, wherein the anticancer agent is selected from the group comprising: docetaxel, cisplatin, gemcitabine, and combinations of cisplatin and gemcitabine.

Other anticancer agents that may be used in combination with the anti-AMHRII antibodies encompass paclitaxel or platinum salts (such as oxaliplatin, cisplatin, and carboplatin).

The anticancer agent may also be selected from chemotherapeutic agents other than platinum salts, small molecules, monoclonal antibodies or other anti-angiogenic peptides.

Chemotherapeutic agents other than platinum salts include intercalators (blocking DNA replication and transcription), such as anthracyclines (doxorubicin, pegylated liposomal doxorubicin), topoisomerase inhibitors (camptothecins and derivatives: karenitecin, topotecan, irinotecan), or SJG-136, inhibitors of histone deacetylases (vorinostat, belistat, valproic acid), alkylating agents (bendamustine, meglumine, temozolomide), antimitotic plant alkaloids (such as taxanes (docetaxel, paclitaxel)), vinca alkaloids (vinorelbine), epothilones (ZK-epothilones, saprepitudes), antimetabolites (gemcitabine, elgilabine, capecitabine), spindle Kinesin (KSP) inhibitors (I Ping Si), trabectedin or Obezocine (combretastatin A-4 derivatives).

Among the small molecules are poly (ADP-ribose) polymerase (PARP) inhibitors: olaparib, ai Ni Pa, uliptinib, lu Kapa Ni, CEP-9722, MK-4827, BMN-673, kinase inhibitors such as Tyrosine Kinase Inhibitors (TKI), among which mention may be made of anti-VEGFR molecules (sorafenib, sunitinib, cilnidinib, vandetanib, pazopanib, BIBF 1120, sema Sha Ni, cabitinib, motaflavinib), anti-HER 2/EGFR molecules (erlotinib, gefitinib), anti-PDGFR molecules (imatinib, BIBF 1120), anti-FGFR molecules (BIBF 1120), aurora kinase/tyrosine kinase inhibitors (ENMD-2076), src/Abl kinase inhibitors (Sakatinib), or also perafaxine, terlazide (mTOR inhibitors), arvoxetine (cyclin-dependent kinase inhibitors), volatin 1 (checkpoint inhibitors of the PLK1 (Potentilla-like kinase 1) protein), 2606368 (kinase 1 (k 1), anti-PDGFR molecules (imatinib, BIBF 1120), anti-FGFR molecules (BIBF 1120), aurora kinase inhibitors (ENMD-2076), src/Abl kinase inhibitors (GmbH), etc., inhibitors, and HIV-18-factor inhibitors.

Among the antibodies, mention may be made of anti-VEGF: bevacizumab, anti-VEGFR: ramucirumab, anti-HER 2/EGFR: trastuzumab, pertuzumab, cetuximab, panitumumab, MGAH, matuzumab, anti-PDGFR alpha: IMC-3G3, antifolate receptor: faraday group mab, anti-CD 27: CDX-1127, anti-CD 56: BB-10901, anti-CD 105: TRC105, anti-CD 276: MGA271, anti AGS-8: AGS-8M4, anti-DRS: TRA-8, anti-HB-EGF: KHK2866, anti-mesothelin: amatuximab, BAY 94-9343 (immunotoxin), cetuximab (EpCAM/CD 3 bispecific antibody), anti-IL 2R: daclizumab, anti-IGF-1R: ganitumumab, anti-CTLA-4: iprimma, anti-PD 1: nivolumab and pembrolizumab, anti-CD 47: weissman B6H12 and Hu5F9, novimmune 5A3M3,INHIBRX 2A1,Frazier VxP037-01LC1 antibodies, anti-lewis Y: hu3S193, SGN-15 (immunotoxin), anti-CAl 25: ago Fu Shan anti, anti-HGF: rituximab, anti-IL 6: steuximab, anti-TR 2: tigetuzumab, anti- α5β1 integrin: wo Luo Cycloximab, anti-HB-EGF: KHK2866. The anti-angiogenic peptide antibody is selected from AMG 386 and CVX-241.

More specifically, described herein are pharmaceutical compositions comprising an anticancer agent and an antibody that binds AMHR-II as active ingredients in combination with a pharmaceutically acceptable vehicle, wherein the anticancer agent is selected from the group comprising: docetaxel, cisplatin, gemcitabine, and combinations of cisplatin and gemcitabine.

Even more particularly, described herein are pharmaceutical compositions comprising an anticancer agent and an antibody that binds AMHR-II as active ingredients in combination with a pharmaceutically acceptable vehicle, wherein the mutated humanized monoclonal antibody referred to herein as 3C23K and the anticancer agent are selected from the group comprising: docetaxel, cisplatin, gemcitabine, and combinations of cisplatin and gemcitabine.

In a particular aspect, described herein are pharmaceutical compositions in formulations intended for administration by intravenous or intraperitoneal routes comprising an anticancer agent as an active ingredient and an antibody that binds AMHR-II in combination with a pharmaceutically acceptable vehicle.

In another particular aspect, the invention relates to the use of a composition comprising an anti-cancer agent and an antibody that binds AMHR-II as a pharmaceutical product in a formulation intended for administration by intravenous or intraperitoneal route in the prevention or treatment of lung cancer.

In another particular aspect, the invention relates to the use of a composition comprising an anti-cancer agent and an antibody that binds AMHR-II, the monoclonal antibody and the anti-cancer agent being intended for separate, simultaneous or sequential administration, as a pharmaceutical product in the prevention or treatment of lung cancer.

The antibody and anticancer agent may be combined within the same pharmaceutical composition, or may be used in the form of separate pharmaceutical compositions administered simultaneously or sequentially. In particular, the products may be applied separately, i.e. simultaneously or independently, for example with time intervals.

More specifically, the present invention relates to the use of a composition comprising an anti-cancer agent and an antibody that binds AMHR-II, wherein the antibody and the anti-cancer agent are combined within the same pharmaceutical composition, as a pharmaceutical product in the prevention or treatment of lung cancer.

According to another particular aspect, the present invention relates to the use of a composition comprising an anti-cancer agent and an antibody that binds AMHR-II as a pharmaceutical product in the prevention or treatment of lung cancer, wherein the therapeutically effective amount of the anti-AMHRII antibody administered to a patient ranges from about 0.07mg to about 35 mg, preferably from about 0.7mg to about 7000mg, preferably from about 0.7mg to about 1400mg, preferably from about 0.7mg to about 700mg and more preferably from about 0.7mg to about 70mg.

According to another particular aspect, the present invention relates to the use of a composition comprising an anticancer agent and an antibody that binds AMHR-II as a pharmaceutical product in the prevention or treatment of lung cancer, wherein the therapeutically effective amount of the anticancer agent administered to a patient ranges from about 10mg to about 700mg, preferably ranging from about 20mg to about 350mg and preferably about 110mg.

According to another particular aspect, the present invention relates to the use of a composition comprising an anti-cancer agent and an antibody that binds AMHR-II as a pharmaceutical product in the prevention or treatment of lung cancer, wherein the therapeutically effective amount of the antibody administered to a patient is about 70mg and the dose of the anti-cancer agent administered to the patient is about 110mg.

In a preferred embodiment, the dose of the anticancer agent (in particular docetaxel or a combination of cisplatin and gemcitabine) ranges from about 0.01mg/kg to about 500mg/kg, such as 0.1mg/kg to 300mg/kg, or about 0.1mg to 20g per day.

Alternatively, a higher initial loading dose may be administered followed by one or more lower doses. In another variation, a less high initial loading dose may also be administered, followed by one or more higher doses.

In particular embodiments, the anti-AMHRII antibody and anti-cancer agent may be used in an antibody/anti-cancer agent weight ratio ranging from about 10/1 to about 0.01/1, particularly from about 10/1 to about 0.05/1, or from about 5/1 to about 0.1/1.

Illustratively, the anti-AMHRII antibody and docetaxel may be used at a weight ratio of antibody/docetaxel of 1/1, as shown in the examples herein.

Still illustratively, the anti-AMHRII antibody and cisplatin may be used at an antibody/cisplatin weight ratio of 4/1, as shown by the examples herein.

Still illustratively, the anti-AMHRII antibody and gemcitabine may be used at an antibody/gemcitabine weight ratio of 0.2/1, as shown in the examples herein.

The invention also describes a product comprising an antibody binding to human anti-mullerian hormone type II receptor (AMHR-II) and an anti-cancer agent in the form of a combined preparation for simultaneous, sequential or separate use as a pharmaceutical product intended for the prevention or treatment of lung cancer expressing AMHRII.

AMHRII binders as disclosed herein, and in particular anti-AMHRII antibodies as disclosed herein, can be administered in a variety of ways, including oral administration, subcutaneous administration, and intravenous administration.

The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow down, preferably stop to some extent) infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down, preferably stop to some extent) tumor metastasis; inhibit tumor growth to some extent; and/or to some extent, alleviate one or more symptoms associated with the disorder. To some extent, the drug may prevent growth and/or kill existing cancer cells, it may inhibit cell growth and/or be cytotoxic. For cancer therapy, in vivo efficacy may be measured by, for example, assessing the duration of survival, the duration of Progression Free Survival (PFS), response Rate (RR), the duration of response, and/or quality of life.

Therapeutic formulations of reagents (e.g., antibodies) for use according to the invention in the form of stored, lyophilized formulations or aqueous solutions are prepared by mixing antibodies of the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (remington's Pharmaceutical Sciences th edition, osol, editions (1980)). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethyldiammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zn-protein complexes); and/or nonionic surfactants such as TWEEN ^TM、PLURONICS^TM or polyethylene glycol (PEG).

The active ingredient may also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly- (methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in remington's Pharmaceutical Sciences, 16 th edition, osol, a. Edit (1980).

Formulations for in vivo administration may be sterile. This is easily achieved by filtration through sterile filtration membranes.

The pharmaceutical compositions as described herein may be administered by any suitable route of administration, for example by parenteral, oral, sublingual, vaginal, rectal or transdermal route, preferably by intravenous, subcutaneous or intradermal injection. Intramuscular, intraperitoneal, intrasynovial, intrathecal or intratumoral injection are also possible. The injection may be in the form of bolus injection or by continuous infusion. When the antibody composition and the anticancer agent composition are administered separately, these compositions may be in the same or different administration forms.

Formulations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) or injectable organic esters (such as ethyl oleate). Aqueous vehicles include water, alcohol/water solutions and emulsions or suspensions.

The pharmaceutical compositions as described herein advantageously comprise one or more pharmaceutically acceptable excipients or vehicles. Saline, physiological solutions, isotonic solutions, buffer solutions, etc. which are compatible with pharmaceutical use and known to the person skilled in the art may be mentioned, for example. The composition may contain one or more agents or vehicles selected from dispersants, solubilisers, stabilisers, preservatives and the like. Agents or vehicles useful in the formulation (liquid and/or injectable and/or solid) are in particular methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia and the like. The compositions may be formulated in the form of injectable suspensions, gels, oils, tablets, suppositories, powders, hard gelatin capsules, soft capsules and the like.

According to a particular aspect, the present invention relates to a pharmaceutical composition comprising as active ingredients an anti-cancer agent and an anti-AMHRII antibody in combination with a pharmaceutically acceptable vehicle, wherein the therapeutically effective amount of the antibody administered to the patient ranges from about 0.07mg to about 35000mg, preferably from about 0.7mg to about 7000mg, preferably from about 0.7mg to about 1400mg, preferably from about 0.7mg to about 700mg and more preferably from about 0.7mg to about 70mg.

The dosage of the active ingredient depends inter alia on the method of administration and is readily determinable by the person skilled in the art. The therapeutically effective amount (unit dose) of the antibody may vary from 0.01mg/kg to 500mg/kg, preferably from 0.1mg/kg to 100mg/kg, preferably from 0.1mg/kg to 20mg/kg, preferably from 0.1mg/kg to 10mg/kg and more preferably from 1mg/kg to 10mg/kg, for one or more administrations per week for weeks or months. Thus, an effective unit dose can be easily deduced from the dose calculated for an "average" patient weighing 70 kg.

According to another specific aspect, the present invention relates to a pharmaceutical composition comprising as active ingredients an anti-cancer agent and an anti-AMHRII antibody in combination with a pharmaceutically acceptable vehicle, wherein the therapeutically effective amount of the anti-cancer agent administered to the patient is in the range of about 10mg to about 700mg, preferably in the range of about 20mg to about 350mg, and preferably about 110mg.

The dosage of the anticancer agent depends particularly on the administration method and is easily determined by one skilled in the art. In one or more weekly administrations lasting weeks or months, the therapeutically effective amount (unit dose) may vary from 0.2mg/m ² to 10g/m ², preferably from 0.2mg/m ² to 1g/m ², preferably from 2mg/m ² to 1g/m ², preferably from 20mg/m ² to 1g/m ² and more preferably from 20mg/m ² to 0.5g/m ². Thus, an effective unit dose can be derived from the dose calculated for an "average" patient having a body surface area of about 1.8m ².

According to an even more specific aspect, the present invention relates to a pharmaceutical composition comprising as active ingredients an anti-cancer agent and an anti-AMHRII antibody in combination with a pharmaceutically acceptable vehicle, wherein the therapeutically effective amount of the anti-cancer agent administered to the patient is about 110mg and the therapeutically effective amount of the antibody administered to the patient is about 70mg.

The invention also describes the use of a composition comprising an anti-cancer agent and an anti-AMHRII antibody that binds to the human anti-mullerian hormone type II receptor (AMHR-II) as a pharmaceutical product in the prevention or treatment of lung cancer that expresses AMHRII.

The invention is further illustrated by the following examples, but is not in any way limited to the following examples.

Examples

Example 1: differential AMHRII Gene expression and AMHRII protein expression

A. Materials and methods

A.1. cell lines and cultures

The COV434 WT cell line (ECACC No. 07071909) was maintained in DMEM/GlutaMax (Gibco) supplemented with 10% FBS, penicillin 100U/ml and streptomycin 100. Mu.g/ml. Geneticin (Gibco) was added at 400 μg/ml for the COV434 MIRII transfected cell line. Erythroleukemia K562 cell lineCCL-243 ^TM) was suspended in IMDM medium (Sigma Aldrich) supplemented with 10% fbs and penicillin/streptomycin and maintained in T75 flasks at a density of 1x 10 ⁵ to 1x 10 ⁶ cells/ml. OV90 cell line (/ >)CRL-11732 ^TM, ovarian serous adenocarcinoma) was cultured in a 1:1 mixture of MCDB 105 medium (Sigma Aldrich) with a final concentration of 1.5g/l sodium bicarbonate and medium 199 (Sigma Aldrich) (supplemented with 15% FBS and penicillin/streptomycin) with a final concentration of 2.2g/l sodium bicarbonate. NCI-H295R cell line (adrenocortical carcinoma,CRL-2128 ^TM) was maintained in DMEM:F12 medium (Sigma Aldrich) supplemented with iTS ⁺ Premix (Corning), 2.5% Nu-Serum (Falcon) and penicillin/streptomycin. Cells were grown at 37 ℃ in a humid atmosphere with 8% co ₂ and the medium was changed once or twice a week depending on the cell line.

A.2. relative quantification of AMHR2 mRNA by RT-qPCR

Extracting RNA according to manufacturer's instructionsPlus RNA purification kit (Ambion) total RNA from 1x10 ⁶ to 5x10 ⁶ cell pellet was prepared. Briefly, after phenol/chloroform extraction, RNA from lysed cells was adsorbed onto a silica matrix, treated with DNase, then washed with 30. Mu.l RNase-free water and eluted. RNA concentration and quality were assessed using a spectrophotometer (NanoDrop, siemens sciences (ThermoFisher Scientific)).

RNA (1. Mu.g) was reverse transcribed using Maxima H Minus first strand cDNA synthesis kit (Ambion) and oligo-dT primers by the following steps: incubation at 25℃for 10min for priming and at 50℃for 15min for reverse transcription followed by incubation at 85℃for 5min for reverse transcriptase inactivation.

Quantitative PCR was performed in LIGHT CYCLER (Roche) in 96 well microwell plates using Luminaris Color HIGREEN QPCR MASTER Mix (Ambion) with a final volume of 20 μl. The following primers were used: for AMHR2, forward 5'-TCTGGATGGCACTGGTGCTG-3' (SEQ ID No. 71) and reverse 5'-AGCAGGGCCAAGATGATGCT-3' (SEQ ID No. 72); for TBP, forward direction 5'-TGCACAGGAGCCAAGAGTGAA-3' (SEQ ID NO. 73) and reverse direction 5'-CACATCACAGCTCCCCACCA-3' (SEQ ID NO. 74). Amplification was performed using cDNA template (100 ng equivalent RNA) and following protocol: UDG pretreatment at 50℃for 2min, denaturation at 95℃for 10min, followed by 15s at 95℃/30 s at 60℃/30 s at 70℃for 40 cycles. Melting curve analysis was performed at the end of each experiment to control the absence of genomic DNA and dimer primers. Each cDNA sample and control ("no template sample" and "no reverse transcript RNA") were tested in duplicate. The average of the cycle threshold (Ct) was calculated and AMHR2 Relative Quantification (RQ) was expressed as 2 ^-△△Ct, where ΔΔct= Δct _{Sample of} -△Ct _{Calibration material} and Δct = Ct _AMHR2-Ct_TBP. HCT116 samples were used as calibrator and TBP was used as housekeeping gene for normalization.

Table 2 below depicts AMHRII expression levels in the cell lines tested using the Q-PCR method described above.

TABLE 2

A.3. evaluation of membrane AMHR2 expression by flow cytometry analysis

For Fluorescence Activated Cell Sorting (FACS) analysis, 4X 10 ⁵ cells were incubated with 25. Mu.g/ml 3C23K for 30min at 4 ℃. After washing with PBS-BSA2%, primary antibodies were detected by anti-species secondary antibodies conjugated to fluorophores. 3C23K was detected by anti-human F (ab') ₂ conjugated to phycoerythrin (1:1000, beckman-Coulter, IM 0550). After washing with PBS, FACS analysis of resuspended cells was achieved in the FL2 channel of BD Accuri ^TM C6 flow cytometer (BD Bioscience).

B. Results

The results are depicted in fig. 2. The results showed that although the COV434-WT cell line had a significant membrane expression level of human AMHRII protein, the recombinant cell line COV434-WT (AMHRII gene expression level was about 3% measured for cell line NCI-H295R).

These results indicate that there is no correlation at all between AMHRII gene expression and membrane AMHRII protein expression.

Example 2: AMHRII expression in lung cancer (human tumor samples)

A. Materials and methods

A.1. Target object

Immunohistochemical studies for detection of human cancer cell xenografts (PDX) in mice expressing anti-mullerian hormone type 2 receptor (AMHR 2) using biotinylated 3C23K monoclonal antibody.

A.2. Scheme and method

Cell line: immobilized in formaldehyde acetate (AFA), with a cell mass architecture.

-Human tumor: external samples were fixed in formalin and slides from curie institute were fixed in AFA.

Immunohistochemical (IHC) techniques are possible after dewaxing the sample and exposing it to pH9 (microwave EZ Retriever 15 'at 90 ℃ and then cooling in 20').

Anti-mullerian hormone type II receptor detection revealed by immunoperoxidase technology and DAB chromogenic substrate.

After blocking the endogenous peroxidase activity, the slides were incubated with diluted biotinylated primary antibody (1/800,8. Mu.g/mL) for 90 min at room temperature. The tissue sections were then washed with PBS and incubated with avidin/biotin ABC vector complex for 30 min. The immunoreactive signal was detected using DAB substrate solution (dab+substrate buffer/liquid dab+chromogen, 10 min incubation). Finally, sections were lightly counterstained with mel's hematoxylin (ril improvement).

Negative controls were obtained by replacing primary antibodies with isotype control immunoglobulin (R565) or antibody diluent alone (negative buffer control) in an immunohistochemical staining procedure.

Positive controls were obtained by using AMHR2 transfected COV434 cells and human granulosa cell tumor samples.

After treatment, the slices are observed digitally via PHILIPS IMS. All samples were scored independently by 2 pathologists.

The positioning of the markers is described in detail: cytoplasm and/or membrane.

-Classifying the intensities as clear brown markers of tumor cell membranes and/or cytoplasm by the following scoring system: the intensity of the mark is defined as: 0 (negative), 1 (weak), 2 (medium), and 3 (strong), as shown by COV434 positive control).

Frequency is defined as the percentage of AMHRII expressing cells. Necrotic areas were excluded from the analysis. Total histological scores were established by accumulating membrane expression and cytoplasmic expression using the frequency x intensity score averages (0 to 3).

-Keeping all slides well.

B. Results

Table 3 also depicts the results of AMHRII membrane expression of various primary human lung cancer cells, wherein AMHRII expression scores represent a set of different lung cancer samples.

Table 3: AMHRII expression of human lung cancer tissue samples

Tumor type	Percentage of positive samples	Number of test samples
			SCLC	0％	2
NSCLC (neuroendocrine)	1.2％	78
			NSCLC (acinus type)	0％	2
NSCLC (epidermoid)	100％	4
			NSCLC (squamous cell carcinoma)	35％	14
NSCLC (adenocarcinoma)	45.8％	24
			NSCLC (big cell)	33％	9

The results show that AMHRII is expressed at the cell surface in a plurality of human lung cancer samples, particularly tumor samples of NSCLC origin, more precisely tumor samples derived from patients suffering from NSCLC selected from the group consisting of: epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC and squamous cell carcinoma NSCLC, and neuroendocrine NSCLC.

Example 3: AMHRII expression in lung cancer

A. Materials and methods

A.1. Target object

Studies of human lung cancer cells from patient-derived xenografts (PDX) or from fresh human tumor samples were initiated for detection of anti-mullerian hormone type 2 receptor (AMHR 2) expression using biotinylated 3C23K monoclonal antibody.

A.2. AMHRII Membrane expression analysis by flow cytometry

Preparation of cells for analysis

Tissue was dissected within 1h of surgery, cut into 1-mm2 fragments, and washed in RPMI containing penicillin (10%), streptomycin (10%) and gentamicin (0.1 mg/mL; sigma Aldrich).

The tissue fragments were digested with collagenase and DNase (2 mg/mL; sigma Aldrich) for 2 to 4 hours with rapid shaking at 37 ℃.

Removing mucus and large debris by filtration through a 40-lm cell strainer.

Viable cells were obtained by Ficoll gradient centrifugation.

The AMHRII binding sites on resuspended tumor cells were quantified using The QuantumTM Simply Cellular (Bangs Laboratory) according to the manufacturer's instructions:

Briefly, four microbead populations labeled with different calibrated amounts of mouse anti-human IgG specific for the Fc portion of human IgG antibodies were stained with AlexaFluor488 conjugated anti AMHRII C23K. In FACS tubes, one drop of each vial in the kit is added to 50 μl of PBS 1X:

1-bead B (blank)

2-Bead 1+3C23K-AF 10. Mu.g/mL

3-Bead 2+3C23K-AF 10. Mu.g/mL

4-Bead 3+3C23K-AF 10. Mu.g/mL

5-Bead 4+3C23K-AF 10. Mu.g/mL (the concentration can be increased to 25. Mu.g/mL if necessary).

Each bead population was combined with a different amount of AlexaFluor488 conjugated anti AMHRII C23K, yielding corresponding fluorescence intensities, which were analyzed on FACS Canto II cytometer (BD).

-Generating a calibration curve by plotting the average fluorescence intensity of each bead population against its designated Antibody Binding Capacity (ABC).

Cells were stained typically in 1.5ml Ai Bende tubes.

All centrifugation steps are carried out at 4 ℃.

All incubation steps were performed at 4 ℃ to avoid antibody internalization.

350 Ten thousand cells (trypsinized COV 434-MIRII or freshly dissociated tumor cells) were centrifuged at 200g to 300g for 5 min and washed once with PBS (500. Mu.l per tube).

Wash with ice-cold PBS/2% FBS (200 g to 300g for 3 min), and re-suspend in 700. Mu.l PBS 1X and dispense 100. Mu.l through FACS tubes, conditions are described in Table 4 below:

TABLE 4 Table 4

Incubation with antibody 3C23K-AF488 in PBS/1% FBS at 4℃for 30min

Washing twice in PBS/2% BSA (200 g to 300g for 3 min)

Washing twice in PBS (200 g to 300g for 3 min)

300. Mu.l to 400. Mu.l PBS was added and analyzed on FACS as soon as possible

This protocol does not include any fixation step for extracellular staining to preserve membrane integrity. Thus, only the membrane AMHRII was detected.

A.3. immunohistochemistry: scheme and method

Cell line: immobilized in formaldehyde acetate (AFA), with a cell mass architecture.

-Human tumor: external samples were fixed in formalin and slides from curie institute were fixed in AFA.

Immunohistochemical (IHC) techniques are possible after dewaxing the sample and exposing it to pH9 (microwave EZ Retriever 15 'at 90 ℃ and then cooling in 20').

Anti-mullerian hormone type II receptor detection revealed by immunoperoxidase technology and DAB chromogenic substrate.

After blocking the endogenous peroxidase activity, the slides were incubated with diluted biotinylated primary antibody (1/800,8. Mu.g/mL) for 90 min at room temperature. The tissue sections were then washed with PBS and incubated with avidin/biotin ABC vector complex for 30 min. The immunoreactive signal was detected using DAB substrate solution (dab+substrate buffer/liquid dab+chromogen, 10 min incubation). Finally, sections were lightly counterstained with mel's hematoxylin (ril improvement).

Negative controls were obtained by replacing primary antibodies with isotype control immunoglobulin (R565) or antibody diluent alone (negative buffer control) in an immunohistochemical staining procedure.

Positive controls were obtained by using AMHR2 transfected COV434 cells and human granulosa cell tumor samples.

After treatment, the slices are observed digitally via PHILIPS IMS. All samples were scored independently by 2 pathologists.

The positioning of the markers is described in detail: cytoplasm and/or membrane.

-Classifying the intensities as clear brown markers of tumor cell membranes and/or cytoplasm by the following scoring system: the intensity of the mark is defined as: 0 (negative), 1 (weak), 2 (medium), and 3 (strong), as shown by COV434 positive control).

Frequency is defined as the percentage of AMHRII expressing cells. Necrotic areas were excluded from the analysis. Total histological scores were established by accumulating membrane expression and cytoplasmic expression using the frequency x intensity score averages (0 to 3).

-Keeping all slides well.

B. Results

A) Control

Negative control and isotype control are not reactive to tumor cells.

Positive control samples (amplified COV434 AMHRII) showed diffuse immunostaining of cells (intensity score: 3). The markers were uniform for cytoplasmic localization and membrane localization (frequency score: 100%).

Positive granulosa cell control samples showed strong immunostaining of tumor cells (intensity score: 3). The markers were uniform for cytoplasmic localization and membrane localization (frequency score: 100%).

B) AMHRII expression of patient-derived xenograft (PDX) samples as assessed by IHC.

It is important to note that when samples were fixed in formalin, membrane expression of AMHR2 appeared to be underestimated compared to samples treated in AFA.

The results of AMHRII membrane expression of various human tumors xenografted in mice are depicted in table 5, wherein AMHRII expression scores represent a set of different cancer cell types.

Table 5 below summarizes some of the results of AMHRII expression of human tumor xenografts.

Table 5: AMHRII expression in human tumor xenografts

Tumor type	Percentage of positive PDX	Number of PDX tested
			SCLC	0％	13
NSCLC (unspecified subtype)	15.4％	13
			NSCLC (epidermoid)	26.9％	26
NSCLC (adenocarcinoma)	7.7％	39
			NSCLC (big cell)	40％	10

The results indicate that AMHRII is expressed at the cell surface in a variety of human lung cancer xenografts, particularly tumor samples of NSCLC origin, more precisely tumor samples derived from patients with NSCLC selected from the group consisting of: epidermoid NSCLC, adenocarcinoma NSCLC, large cell NSCLC and some NSCLCs of which subtypes have not been identified.

C) AMHRII expression of patient-derived xenograft (PDX) samples as assessed by flow cytometry (FACS)

The results depicted in fig. 3A-3E demonstrate that AMHRII is expressed at the tumor cell membrane of xenografts derived from lung cancer patients, independent of the type of lung cancer considered. The results depicted in fig. 3A-3E demonstrate that AMHRII membrane protein expression was found in squamous cell lung cancer (fig. 3A, 3C, 3D), large cell lung cancer (fig. 3B), and polymorphic cell lung cancer (fig. 3E).

Furthermore, for the same lung cancer cells, (i) AMHRII number per cell and (ii) percentage of AMHRII cancer cells were measured in the same test samples. The results are depicted in table 6 below.

Table 6: FACS analysis of AMHRII expression in human lung cancer cells obtained from patient-derived xenografts

In table 6, AMHRII expression in each tumor sample was assessed by (i) determining the average number of AMHRII proteins present at the tumor cell membrane and (ii) determining the percentage of membrane AMHRII positive cells in the tumor sample. The left column of table 6 shows an indication of setting the corresponding tumor sample to "positive" or "negative". The indication "positive" means that the tumor cells of the lung cancer patient are significantly expressed AMHRII at their membrane. The indication "negative" means that AMHRII was not significantly detected at the tumor cell membrane.

The results in table 6 show that all tumor samples expressed membrane AMHRII despite the various expression levels.

D) AMHRII expression of fresh human tumor samples as assessed by flow cytometry (FACS)

The results depicted in fig. 3F and 3G indicate that AMHRII is expressed at the tumor cell membrane derived from surgically resected human NSCLC (fig. 3G), while AMHRII is not expressed at the cell membrane derived from healthy margin produced from the same patient (fig. 3F).

E) Conclusion(s)

AMHR2 protein expression has been confirmed in a lung cancer PDX model positive for AMHR2 transcription. These PDX changed from lung IC8LC10 cancer and lung SC131 cancer. The expression level was moderate but significant, characterized by a total score of 1 to 1.5. These data indicate that AMHR2 can be expressed in all but gynaecological cancers.

These models may be used in the future to characterize anti-AMHR 2 therapies.

Example 4: in vivo efficacy of anti-AMHRII antibodies against AMHRII expressing lung cancer

1. Summary of the objects

To analyze the antitumor efficacy of the test compound GM102 (also referred to herein as 3C23K antibody) plus Ma Ma buss (Gamamab) as a single agent or in combination with docetaxel or combined cisplatin/gemcitabine in SC131 patient-derived non-small cell lung xenograft models developed in immunodeficient female mice.

2. Method of

Fifty-four (54) mice with SC131 tumors (P22.1.3/0) grown subcutaneously 62.5mm ³ to 220.5mm ³ were assigned to treatment when the average tumor volume and median tumor volume reached 130.76mm ³ and 126.00mm ³, respectively.

Efficacy study XTS-1526 included 6 groups of mice, 9 per group:

In group 1, vehicle was administered at 5ml/kg, i.v.2qwkx3;

in group 2, GM102 was administered at 20mg/kg, i.v.2qwk x 3;

in group 3 docetaxel was administered once at a slow i.v. at D0 at 20 mg/kg;

In group 4, GM102 was administered at 20mg/kg, i.v.2qwk x1 or 2 in combination with docetaxel administered once at D0 slowly i.v. at 20 mg/kg;

in group 5, cisplatin was administered at 5mg/kg in combination with gemcitabine administered at 100mg/kg, both i.p. qwk x2 or 3;

In group 6, GM102 was administered at 20mg/kg, i.v.2qwkx1 or 2, together with a combination of cisplatin administered at 5mg/kg and gemcitabine administered at 100mg/kg (both i.p.qwkx1 or 2).

Of the non-enrolled efficacy study mice, 2 groups were tested, each group comprising 8 mice:

In group 7, GM102 was administered at 20mg/kg, i.v.2qwkx3 in combination with cisplatin administered at 5mg/kg, i.p.qwkx3;

In group 8, GM102 was administered at 20mg/kg, i.v.2qwkx3 in combination with gemcitabine administered at 100mg/kg, i.p.qwkx3.

During the experiment, tumors were measured and mice were weighed 3 times per week. Fresh tumor samples were collected from 3 mice per group, and no additional dose was applied to the quick frozen tissue and formalin-fixed samples at either D28 (for 2 nd and 3 rd) or D31 (for 1 st). Only the quick frozen tissue is advanced forward for subsequent analysis. Formalin fixed samples were discarded after sampling.

3. Purpose of investigation

The experiments described in this report were aimed at determining the antitumor efficacy of a test compound (encoded as GM 102) of (Gamamab) alone or in combination with docetaxel or combined cisplatin/gemcitabine in a non-small cell lung xenograft model derived from SC131 patients.

Test item: GM102 (also referred to herein as 3C 23K)

The anti-AMHR 2 product GM102 is a humanized mAb directed against the anti-muller hormone receptor (AMHR 2), also known as the muller inhibitor receptor II (MISRII). AMHR2 is present at the level of internal female sex organ precursors (Miao Leguan) during the uterus and is limited to the ovary (granulosa cells) and testis (testicular interstitial cells) during adulthood. AMHR2 is also expressed in about 65% of gynaecological cancers such as ovary and endometrium (Bakkum JN,Gynecol Oncol,2007;Sahli I,Biochem,2004;Anttonen M,Lab Invest,2011;Song JY,Int J.Oncol,2009).

GM102 antibodies have been shown to exhibit anti-tumor efficacy in a mouse xenograft model using AMHR2 transfected human tumor cell lines. This efficacy has been shown to depend on participation of immune effector cells triggered by antibodies that enable optimization at the tumor level. Furthermore, GM102 efficacy has been shown to have a synergistic effect with carboplatin and paclitaxel (the primary chemotherapeutic used in ovarian cancer) (Jacquet a., CANCER RES, 2012).

Human tumor xenograft model

Various histological-derived human tumor samples were obtained with informed consent from cancer center-treated patients and established as transplantable xenografts in immunodeficient mice. The implanted sample is residual material from the primary tumor or metastasis obtained either before or after treatment. These patient-derived xenograft (PDX) models can be established without prior in vitro culture, and histological, cytogenetic, genetic, and other biological markers and their responses to standard of care (SOC) therapies have been studied.

SC131 tumor model was derived from skin metastases of non-small cell lung cancer with mutated EGFR (R451F) and Kras (G12V), wild-type TP53 and PTEN.

SC131 was a low responder to both docetaxel and cisplatin/gemcitabine combinations and no responder to other test agents (data obtained from swiss nude mice).

The SC131 tumor model takes approximately 17 days to obtain a maximum tumor of 60mm ³ to 200mm ³ and 35 to 40 days to reach 2000mm ³ from the day of implantation.

SC131 shows cachexia properties.

4. Material

4.1. Animal and maintenance conditions

Distant athymic (nu/nu) female mice weighing 18 to 25 g (HSD: athymic nude-Foxn 1 ^nu) (ENVIGO, ganna, france) were assigned to acclimation in animal facilities to obtain food and water ad libitum for at least 6 days prior to handling (Table 7).

Table 7: animal characteristics

4.2. Animal welfare statement

Permission to use animals in CERFE facilities was obtained from the department of veterinary services of the French agriculture and fishery (the direct DES SERVICES V e rinaires, minist e de l' Agriculture et de la P e che) (protocol No. B-91-228-107). Animal care and containment comply with french regulatory regulations concerning the protection of laboratory animals.

All experiments were conducted in accordance with the laws concerning laboratory animal protection and in accordance with the currently available vertebrate experimental license issued by the department of Agriculture and fishery of france (FRENCH MINISTRY for agricultural AND FISHERIES) to Guillaume Lang (No. a-75-1927, date: 2012, 4, 15, and expiration date: 5).

4.3. Animal feeding

Mice were housed in groups of up to 7 animals during the acclimation period and in groups of up to 6 animals during the experimental period. Mice were housed in individual ventilated cages (ivcs) (mm 213W x 362D x 185H, allen) of Polysulfone (PSU) plastic with a sterilized and dust-free bedding core. Both food and water have been sterilized. Animals were housed in a bright-dark cycle (14 hours artificial light circadian cycle) and controlled room temperature and humidity.

Environmental conditions were monitored as required and data was kept in a central animal containment archive (CENTRAL ANIMAL House industries).

4.4. Meal and water supply

Drinking water was provided ad libitum. Throughout the course of the study, each mouse was provided with a complete pellet diet (150-SP-25 type, SAFE) daily. The CERFE location maintains analytical certificates for animal food and water.

4.5. Animal identification

Prior to each experiment, all animals were weighed and identified by means of a unique ear model numbering system.

Each cage is identified with a paper label, indicating: cage number, mouse strain and number, tumor code, date of experiment.

4.6. Test compounds and formulations

PBS 1X vehicle was prepared by diluting PBS 10X (Sigma PBS 10X, #P5493-1L, batch SLBJ 2848) with 1/10 in sterile deionized water. It was stored at 4 ℃ for 30 days for treatment of aliquots and GM102 dilution.

GM102 (3C 23K) was received (4 vials 5ml,10.1 mg/ml) from 7-2016 of a concentrated aliquot (batch LP01[ R18H2-LP01 ]), and stored at 4 ℃. On each dosing day, stock solutions were diluted in cold PBS 1X to obtain 2mg/ml working solution. The solution was kept on ice or at 4 ℃ and protected from light until handled, then the vials were kept at room temperature during injection. The working solution remaining after the treatment is discarded.

10Mg/ml docetaxel @ prior to each administrationCynophenanthrene (Sanofi), batch 6F 255A-due date: month 2018) stock must be diluted 1/5 with 0.9% nacl to obtain a working concentration of 2 mg/ml. The stock solution is stable for one month in dark after being redissolved at 4 ℃.

A0.5 mg/ml stock solution of cisplatin (cisplatin-Teva, lot 15A30 MF-due date: 2017, month 01) was readily available. The solution was kept at room temperature and protected from light until the supplier's expiration date.

40Mg/ml gemcitabineGift (Lilly), lot number C442937D, due date: month 2018) stock must be diluted 1/4 with 0.9% nacl prior to each administration to obtain a working concentration of 10 mg/ml. The stock solution is stabilized for one month after being redissolved and protected from light at 4 ℃.

5. Method of

5.1. Tumor graft model induction

Tumors of the same generation were subcutaneously transplanted onto 3 to 24 mice (donor mice, passage (n-1)). When these tumors reached 700mm ³ to 2000mm ³, donor mice were sacrificed by cervical dislocation and the tumors were aseptically resected and dissected. After removal of the necrotic area, the tumor was cut into pieces measuring about 20mm ³ and transferred to the medium prior to implantation.

Nineteen (89) mice were anesthetized with 100mg/kg ketamine hydrochloride (batch 5D 92-due date: 03-2017, virbac) and 10mg/kg xylazine (batch KP0AX9X, bayer (Bayer)) and then skin was sterilized with chlorhexidine solution, dissected horizontally in the inter-scapular region, and 20mm ³ tumor fragments were placed in subcutaneous tissue. The skin was closed with a clip.

All mice from the same experiment were implanted on the same day.

5.2. Treatment period

In the XTS-1526 efficacy section, 54 mice with SC131 tumors (P22.1.3/0) of 62.5mm ³ to 220.5mm ³ grown subcutaneously were allocated according to tumor volume to give a uniform average tumor volume and median tumor volume for each treatment group. Treatment was randomly assigned to a box containing up to 5 mice and treatment was started 18 days after tumor implantation (60% inclusion rate (with staggered inclusion)). Study was included in a staggered fashion, with 5 mice per group first, and then 4 mice per group after 2 days. The study was terminated 31 days after the start of treatment.

TABLE 8

For the other groups 7 and 8, the tumor sizes were higher and less uniform. Animals were included 32 days after implantation.

TABLE 9

5.3. Tumor measurement and animal observation

Tumor volume was assessed by measuring tumor diameter three times a week during treatment. Use TV (mm ³) = [ length (mm) x width (mm) ² ]/2, where length and width are the longest diameter and shortest diameter of the tumor, respectively.

During the treatment period, all animals were weighed 3 times per week. Adverse reactions to the different treatments were determined as:

for each measurement, the relative weight (RBW) is calculated by dividing the weight by the weight at the beginning of the treatment.

Percent body weight loss (% BWL) =100- (BW _x/BW₀ x 100) in individuals, where BW _x is BW on any day during treatment and BW ₀ is BW on the first day of treatment.

Mice were observed daily for physical appearance, behavior and clinical changes.

All signs of disease were recorded for each animal and any behavioral changes or responses to treatment.

Design of XTS-1526 study

A total of 8 groups were used as summarized in table 9. For groups 1 to 6, each group initially included 9 mice. For groups 7 and 8, each group initially included 8 mice.

In group 1, the vehicle was administered twice weekly by the intravenous (i.v.) route at 5ml/kg for 3 weeks.

In group 2, GM102 was administered twice weekly by the intravenous route at 20mg/kg for 3 weeks.

In group 3, docetaxel was administered once at 20mg/kg by intravenous route at D0.

In group 4, GM102 was administered twice weekly by the intravenous route for 1 or 2 weeks in combination with docetaxel administered once at 20mg/kg by the intravenous route at D0.

In group 5, cisplatin was administered at 5mg/kg in combination with gemcitabine administered at 100mg/kg, each once weekly by the intraperitoneal (i.p.) route for 2 or 3 weeks.

In group 6, GM102 was administered twice weekly by the intraperitoneal route for 1 or 2 weeks at 20mg/kg for 1 or 2 weeks, along with a combination of cisplatin administered at 5mg/kg and gemcitabine administered at 100mg/kg (all once weekly by the intraperitoneal route for 1 or 2 weeks).

In group 7, GM102 was administered twice weekly by the intravenous route for 3 weeks, together with cisplatin administered at 5mg/kg once weekly by the intraperitoneal route for 20 mg/kg.

In group 8, GM102 was administered twice weekly by the intravenous route for 3 weeks, along with gemcitabine administered at 100mg/kg once weekly for 3 weeks by the intraperitoneal route.

All therapeutic doses were adjusted at each injection according to body weight.

Table 10: dose and dose schedule in XTS-1526 efficacy studies

5.5. Behavior in case of weight loss or adverse events

If any side effects are observed on the day of tumor measurement and weight monitoring (three times a week) or weight loss is 15% or more compared to the day of inclusion, the sponsor is notified from the shortest delay in finding side effects/problems.

Then, the following actions are performed:

-stopping the treatment of the animal; if weight loss <10%, treatment is resumed;

DietGel is administered to the whole group in which weight loss is observed And weighing the corresponding animals daily until weight loss is <10%; if weight loss is <10%, then stop DietGelAnd (5) adding.

5.6. Ethical sacrifice criteria

Animals were sacrificed according to the following criteria:

Weight Loss (BWL) of > 20% for 48 consecutive hours (3 measurements) compared to the first day of treatment.

General changes in behaviour or clinical signs.

Tumor volume not less than 2000mm ³.

5.7. Endpoint/study termination

Only mice meeting ethical sacrifice criteria were sacrificed at the appropriate time.

All experimental groups ended at the end of the experimental period.

The endpoints of the experiment were:

a treatment period of-4 weeks, in which,

No follow-up period.

5.8. Blood, tumor and tissue sampling

5.8.1. Tumor sampling

5.8.1.1. Tumor sampling for FFPE

1/2 Tumor treatments were used for FFPE: tumors were fixed in 10% formalin for 24 hours and metastasized in 70% ethanol, then sent to Histalim at the following addresses for paraffin embedding (i.e., 17 [ from major study ] FFPE tumor samples):

The exact sampling time and formalin fixation duration of each tumor sample was recorded.

During the modification 5 editing process, the sponsor decides to discard FFPE samples.

5.8.1.2. Tumor sampling for quick freezing

1/2 Tumor treatments were used for quick freezing: tumors were cut into 3x3x3mm pieces and snap frozen in liquid nitrogen before transfer to-80 ℃ for storage (i.e. 17 [ from main study ] +6 [ from tolergroup 2 study ] snap frozen tumor samples).

The exact sampling time for each tumor sample was recorded.

5.9. Data analysis

5.9.1. Data processing

All raw data is recorded in the appropriate combination in the programming register, stored and processed by the computer system.

Day 0 was considered the first day of treatment. The days of the experiment were then numbered according to this definition.

The recordings are expressed as mean ± standard deviation of the mean (m ± sem).

The average relative body weight curve will be obtained by plotting the average RBW over time for each experimental group. Delta relative body weight (relative body weight of the treated group compared to the control group) will be used for statistical analysis.

Average percent weight loss (% BWL) =100- (average BWx/average BW0 x 100), where BWx is the average BW for any day during treatment, and BW0 is the average BW for the first day of treatment.

Tumor growth curves will be obtained by plotting the average tumor volume (in mm ³) over time for each experimental group. Delta tumor volume (relative tumor volume of treated versus control) was used for statistical analysis.

Individual Tumor Growth Delay (TGD) is calculated as the time (in days) required for an individual tumor to reach 3 to 5 times the initial tumor volume. Median growth delay/group was calculated and reported in the table.

Tumor growth retardation index (TGDI) was calculated as the median growth retardation of the treated group divided by the median growth retardation of the control group.

The percentage ratio between the mean tumor volume of the treated group (T) and the mean tumor volume of the control group (C) was calculated.

Each measurement was statistically analyzed by the (Mann-Whitney) Mann-Whitney nonparametric comparative test. Each treatment group was compared to the control group.

Tumor Stability (TS) is defined as the number of mice exhibiting a constant tumor size in at least 3 consecutive measurements.

Partial tumor regression (PR) is defined as the number of mice that exhibited a tumor size lower than the initial tumor size in at least 3 consecutive measurements.

Complete tumor regression (CR) was defined as the number of mice exhibiting a tumor size of 0mm ³ to 13.5mm ³ in at least 3 consecutive measurements.

Tumor-free survivors (TFS) were defined as the number of complete tumor regressions recorded until the end of the group day.

6. Results

6.1. Tolerability data, clinical observations

The average percent body weight change during treatment is shown in figure 4.

In this study, mice were weighed three times per week during the experiment.

In group 1, vehicle administered at 5ml/kg, i.v.2qwkx3 was well tolerated, but the cachexia effect of the tumor induced a maximum average weight loss of 8.3% on day 16 and 17.6% of the maximum individual weight loss on day 28. No other adverse events were observed, but DietGel was given to animals from the 2 nd inclusion on day 18, day 21, day 25 and day 26 due to cachexia effects of the tumor

In group 2 GM102 dosed at 20mg/kg, i.v.2qwkx3 was well tolerated with a maximum average weight loss of 9.8% on day 14 and a maximum individual weight loss of 16.8% on day 16, corresponding to the cachexia effect of the tumor observed in control group 1. No other adverse events were observed, but DietGel was given to animals from the 2 nd inclusion on day 11, day 16, day 18, from day 21 to day 27 due to cachexia effects of the tumorMice # 27 were found to die on day 27 without any clinical signs.

In group 3, docetaxel administered once at D0 at 20mg/kg, i.v. induced a statistically significant (p <0.01 from day 4) maximum average weight loss of 17.0% on day 16 and 23.8% maximum individual weight loss on day 19 compared to control group 1. No other adverse events were observed, but DietGel were given to the whole group from day 7 to day 27 (for animals from 1 st inclusion until day 31) due to cachexia effects of the tumorDespite DietGel, 4 mice had to be sacrificed before the end of the study.

In group 4, GM102 administered at 20mg/kg, i.v.2qwk x 1 or 2 in combination with docetaxel administered once at D0 at 20mg/kg i.v. induced a statistically significant (p <0.01 from day 4) maximum average body weight loss of 18.1% on day 14 and 24.1% maximum individual body weight loss on day 23 compared to control group 1. No other adverse events were observed, but DietGel were given to the whole group on days 4 and 5, then from day 7 to day 27 due to cachexia effects of the tumorDespite DietGel administrations, 5 mice had to be sacrificed before the end of the study.

In group 5, cisplatin at 5mg/kg (both i.p. qwk x 2 or 3) in combination with gemcitabine at 100mg/kg induced a statistically significant (p <0.01 from day 2) maximum average weight loss of 17.5% compared to control group 1, and a maximum individual weight loss of 30.1% on day 11. DietGel was administered to animals from the 2 nd inclusion on day 2 and day 3 due to the combined toxicity of the compound combination and cachexia effects of tumor growthThen DietGel/>, was given to the whole group on days 4 and 7Then DietGel/>, was given to the whole group from day 9 to day 27(For animals from 1 st inclusion, up to day 31). Despite DietGel administration, 4 mice had to be sacrificed before the end of the study and 1 mouse was found to die on day 12.

In group 6, GM102 administered at 20mg/kg, i.v.2qwkx1 or 2, in combination with cisplatin administered at 5mg/kg and gemcitabine administered at 100mg/kg (both i.p.qwkx1 or 2), induced a significant (p <0.001 from day 2) maximum average weight loss of 21.1% and a maximum individual weight loss of 27.5% on day 11 compared to control group 1. DietGel was administered to animals from the 2 nd inclusion on day 2 and day 3 due to the combined toxicity of the compound combination and cachexia effects of tumor growthThen DietGel is given to the whole group from day 4 to day 27(For animals from 1 st inclusion, up to day 31). Despite DietGel administration, 7 mice had to be sacrificed before the end of the study.

In the other group 7, GM102, administered at 20mg/kg, i.v.2qwkx3 and combined with cachexia effects of tumor growth, together with cisplatin administered at 5mg/kg, i.p.qwkx3, induced a significant maximum average body weight loss of 12.3% on day 18 and 28.9% of the maximum individual body weight loss on day 28. DietGel is administered to animals on days 9 and 11, then from day 13 to day 28 due to the combined toxicity of the combination of compounds and the cachexia effect of tumor growthDespite DietGel administration, 2 mice had to be sacrificed and 1 mouse was found to die before the end of the study. Furthermore, 5 out of 8 mice showed desquamation or/and dry skin from day 8 to the end of the study.

In the other group 8, GM102, administered at 20mg/kg, i.v.2qwk x 3, combined with cachexia effects of tumor growth, together with gemcitabine administered at 100mg/kg, i.p. qwk x 3, induced a significant maximum average body weight loss of 13.4% on day 11 and 26.4% on day 28. Due to the combined toxicity of the combination of compounds and the cachexia effect of tumor growth, dietGel is administered to the animals on days 2 to 4, 7 to 9, 11 and 12, and then 14 to 28Despite DietGel administration, 3 mice had to be sacrificed and 1 mouse was found to die before the end of the study. Furthermore, 6 out of 8 mice showed desquamation or/and dry skin from day 4 to the end of the study.

6.2. Data on antitumor efficacy

The tumor growth curve (average tumor volume over time) is shown in figure 4. The T/C percentage values for each treatment group are presented in table 11 and shown in fig. 5 and 6. Statistical analysis is shown in table 12.

In this study, tumors were measured three times per week during the experiment.

In group 2 GM102 dosed at 20mg/kg, i.v.2qwkx3 did not show any antitumor efficacy on day 16 (end of control group), with tgdi=1.33 and optimal T/c=74.68%.

Docetaxel administered once at D0 at 20mg/kg, i.v. in group 3 showed strong and statistically significant antitumor efficacy on day 16 (end of control group) (by the man-wheatman test, p <0.01 compared to control group 1, then from D7 to D16, p < 0.001), with TGDI >2.71 and optimal T/c=11.00%). Furthermore, transient tumor stabilization in 7 out of 9 mice and transient partial tumor regression in 2 out of 9 mice were observed during the treatment period.

In group 4 GM102 administered at 20mg/kg, i.v.2qwk x 1 or 2 in combination with docetaxel administered once at D0 at 20mg/kg, i.v.2qwk x 1 or 2 showed strong and statistically significant (by the man-huffman test, p <0.01, then D7 to D14, p <0.001 compared to control group 1) antitumor efficacy at D4 at day 16 (group 4 end, n=6) with TGDI >2.71 and optimal T/c=11.34%. In addition, transient tumor stabilization in 6 of 9 mice and transient partial tumor regression in 3 of 9 mice were observed during the treatment period.

In group 5, cisplatin (both i.p. qwk x2 or 3) administered at 5mg/kg in combination with gemcitabine administered at 100mg/kg showed statistically significant (by the mann-huffman test, p <0.01, then D7 to D11, p <0.001 compared to control group 1) antitumor efficacy at day 16 (group 5 end, n=6), with tgdi=2.30 and optimal T/c=27.16%. Furthermore, transient tumor stabilization was observed in 5 out of 9 mice during the treatment period.

In group 6 GM102 administered at 20mg/kg, i.v.2qwkx1 or 2 in combination with cisplatin administered at 5mg/kg and gemcitabine administered at 100mg/kg (both i.p.qwkx1 or 2) showed statistically significant (by the mann-huffman test, p <0.05 at D2, then D4 to D11, p <0.001 compared to control group 1) antitumor efficacy at day 11 (end of group 6, n=7) with tgdi=1.98 and optimal T/c= 33.71%. Furthermore, transient tumor stabilization was observed in 6 out of 9 mice during the treatment period.

In the other groups 7 and 8, some transient tumor stabilization was observed during the treatment, 5 out of 8 mice for the combination GM 102/cisplatin and 6 out of 8 mice for the combination GM 102/gemcitabine, due to the higher average tumor volume at the time of group entry, which was not comparable to control group 1.

7. Conclusion(s)

Results and discussion

The cachexia effect of the SC131 tumor model was higher than expected and resulted in similar weight loss in the vehicle and GM102 treated groups. Thus, GM102 alone can be considered well tolerated.

In another aspect, the toxicity observed in the 4 other groups was due in part to the standard of care docetaxel, cisplatin, and gemcitabine, and induced death of about half of the mice in each group.

GM102 antibody alone induced 25% inhibition of tumor growth, which was not statistically significant, whereas the standard of care group showed strong inhibition of tumor growth. This result was surprising because the model was initially selected based on its membrane AMHRII expression (1+ by IHC score). However, when membrane AMHRII expression on SC131 PDX tumors was assessed concurrently with this study, a decrease in membrane AMHRII expression was noted over several passages (score 0.2+;40% positive cells score 0.5%). This data demonstrates that AMHRII expression is unstable in certain in vitro and in vivo models, and that membrane expression is critical for AMHRII antitumor efficacy.

For the same reason, by combining GM102 with these standards of care, no enhancement of antitumor activity was observed.

Example 5: in vivo efficacy of anti-AMHRII antibodies against AMHRII expressing lung cancer

A. Materials and methods

A.1. AMHRII Membrane expression by tissue immunochemistry

Thus, a method of conjugation to Alexa using488 Anti AMHRII C23K antibody. Then conjugated to Alexa647 Rabbit anti-AF 488 antibody and goat anti-rabbit antibody were amplified in two steps.

Frozen tissue sections were made with a cryostat Leica CMD1950 maintained at-20 ℃. Frozen tissues were fixed on metal disks with OCT compound and once cured they were fixed on disk supports. A7 μm section was achieved and placed on a Superfrost Plus slide (Menzel) On top of this and stored immediately at-20 ℃.

Frozen section slides were rehydrated with PBS1X, then fixed for 10min at-20℃by covering them with 300. Mu.l of cold acetone (VWR Prolabo) and recovered with parafilm to ensure complete recovery of all tissues from the solution. After washing with PBS, the slides were treated with 300 μl of blocking buffer (PBS 1X-BSA 2% -goat serum 10% -Triton X100.1%) for 1 hour in a wet box at room temperature to block non-specific interactions between antibodies and tissue components. 3C23K-AF488 or isotype control R565-AF488 diluted to 10 μg/ml in blocking buffer was applied in a wet box at room temperature for 30min. After 3 washes (3X 10 min) with PBS1X-Triton X100.1%, anti-AF 488 antibody (Invitrogen) (300 μl) diluted 1/500 in blocking buffer was added and incubated for 30min at room temperature. After 3 washes (3X 10 min) with PBS1X-Triton X100.1%, conjugated anti-rabbit antibody AF647 (Invitrogen) (300 μl) diluted 1/500 in blocking buffer was added and incubated for 30min at room temperature. A0.1% wash with PBS1X-Triton X100 (3X 10 min) was achieved, followed by the application of 0.5. Mu.g/ml DAPI (Sigma Aldrich) for 10min. After rinsing with PBS and H ₂ O, slide sections were fixed under a cover slip (24x50mm,Knittel Glass) with one drop (50 μl) of DAKO fluorescent mounting medium, air bubbles were avoided, and stored in the dark at 4deg.C until imaged.

Image acquisition was performed using a fluorescence microscope leica DM5000B equipped with a CoolSnap EZ CCD camera controlled by Metavue software (mei-grain instrument (Molecular Devices)). Image post-processing was performed using ImageJ free software (http:// ImageJ.

A.2. Human lung tumor xenograft

Tumor fragments were obtained from serial passage xenografts of nude mice. After removal from donor mice, tumors were fragmented (3 mm to 4mm border length) and placed in PBS containing 10% penicillin/streptomycin. Recipient animals were anesthetized by inhalation of isoflurane and received unilateral or bilateral tumor grafts subcutaneously in the flank.

LXFE2226 squamous non-small cell lung cancer model tumor xenografts were subcutaneously implanted, one tumor per mouse (NMRI-Foxn 1 ^nu from charles river (CHARLES RIVER). The experiment consisted of two groups of mice, three of which were euthanized on day 15 for detection of membrane AMHRII expression by flow cytometry. The first group was vehicle control and the second group received the study antibody GM102, which was administered intraperitoneally (i.p.) at a dose level of 20mg/kg twice weekly.

Antitumor efficacy was assessed as the minimum T/C value by comparing the Relative Tumor Volume (RTV) in the group for those days that achieved the best efficacy. After a two week dosing-free observation period, the experiment was terminated on day 43.

Study design:

B. Results

In vivo Activity of GM 102 anti-AMHRII antibody against Lung tumor

The tumor growth curve (average tumor volume over time) is shown in figure 7. Tumors were measured three times per week during the experiment.

The results depicted in fig. 7 and 8 show that anti-AMHRII antibody GM102 exhibited strong anti-tumor activity in all treated xenograft animals.

Tumor growth measurements on day 28 showed that anti-AMHRII antibody GM102 resulted in a dramatic decrease in tumor volume (p < 0.001), meaning that anti-AMHRII antibody (i) prevented tumor growth and (ii) effectively caused lysis of tumor cells originally contained in the tumor xenograft.

Thus, the results of example 5 demonstrate that the anti-AMHRII antibody exerts a highly potent anti-tumor effect against lung cancer cells that actually express AMHRII protein at their membrane, irrespective of the expression level of the gene encoding AMHRII.

Sequence listing

<110> Plus Ma Ma Bus pharmaceutical Co

Curie institute

<120> AMHRII-binding Compounds for preventing or treating Lung cancer

<130> PR77729/KLP/CJ

<140> EP17305446.1

<141> 2017-04-14

<150> EP17305446

<151> 2017-04-14

<160> 74

<170> BiSSAP 1.3.2

<210> 1

<211> 318

<212> DNA

<213> Artificial sequence

<223> "3C_23 VL without leader sequence".

<223> "3C_23 VL without leader sequence"

<223> "3C_23 VL without leader sequence"

<223> "3C_23 VL without leader sequence"

<220>

<223> 3C_23 VL "< 223> without a preamble sequence" 3c_23 VL "< 223> without a preamble sequence

"3C_23 VL without preamble" <223> "3C_23 VL without preamble

<220>

<223> Preamble-free 3c_23 VL "< 223 >" preamble-free 3c_23 VL

<220>

<223> 3C_23 VL without leader sequence

<220>

<223> 3C_23 VL without leader sequence

<220>

<221> CDS

<222> 1..318

<400> 1

gac atc cag atg aca cag tcc cca tct acc ctg tct gct tcc gtg gga 48

gat cgg gtg act atc acc tgc aga gca agc tcc tcc gtg agg tac atc 96

gct tgg tac cag cag aag cca gga aag gcc cca aag ctg ctg acc tac 144

cca acc tcc tcc ctg gaa tcc ggg gtg ccc agc aga ttc tca ggc agt 192

ggc tcc ggc acc gaa ttc acc ctg acc atc agc tca ctg cag cct gac 240

gac ttc gca acc tac tac tgt ctg cag tgg agt agc tac cct tgg aca 288

ttc ggc ggc ggc acc aag gtg gag atc aag 318

<210> 2

<211> 106

<212> PRT

<213> Artificial sequence

<223> Synthetic construct

<223> "[ CDS ] 1:318" from SEQ ID NO 1"

<223> Synthetic construct

<223> Synthetic construct

<223> Synthetic construct

<220>

<223> Synthetic construct <223> synthetic

Construct <223> synthetic construct

[ CDS ] 1:318 from SEQ ID NO 1

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 3

<211> 345

<212> DNA

<213> Artificial sequence

<223> "3C_23 VH without leader".

<223> "3C_23 VH without leader sequence"

<223> "3C_23 VH without leader sequence"

<223> "3C_23 VH without leader sequence"

<220>

<223> 3C_23 VH without leader "< 223 >" 3c_23 VH without leader "< 223>

"3C_23 VH without leader sequence" <223> "3C_23 VH without leader sequence

<220>

<223> 3C_23 VH without leader sequence "< 223 >" 3c_23 VH without leader sequence

<220>

<223> 3C_23 VH without leader sequence

<220>

<223> 3C_23 VH without leader sequence

<220>

<221> CDS

<222> 1..345

<400> 3

cag gtg cgg ctg gtg cag agc ggg gcc gag gtg aag aag cct gga gcc 48

tca gtg aag gtg agt tgc aag gcc tcc ggt tac acc ttc acc agc tac 96

cac atc cac tgg gtc aga cag gct ccc ggc cag aga ctg gag tgg atg 144

ggc tgg atc tac cct gga gat gac tcc acc aag tac tcc cag aag ttc 192

cag ggt cgc gtg acc att acc agg gac acc agc gcc tcc act gcc tac 240

atg gag ctg tct tcc ctg aga tct gag gat acc gca gtc tac tac tgt 288

aca cgg ggg gac cgc ttt gct tac tgg ggg cag ggc act ctg gtg acc 336

gtc tcg agc 345

<210> 4

<211> 115

<212> PRT

<213> Artificial sequence

<223> Synthetic construct

<223> "[ CDS ] 1:345 from SEQ ID NO 3 ]"

<223> Synthetic construct

<223> Synthetic construct

<223> Synthetic construct

<220>

<223> Synthetic construct <223> synthetic

Construct <223> synthetic construct

[ CDS ] 1..345 from SEQ ID NO 3

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<400> 4

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 5

<211> 318

<212> DNA

<213> Artificial sequence

<223> "3C_23k VL without leader sequence".

<223> "3C_23K VL without leader sequence"

<223> "3C_23K VL without leader sequence"

<223> "3C_23K VL without leader sequence"

<220>

<223> 3C_23K VL without leader sequence "< 223 >" 3C_23K VL without leader sequence "< 223>

"3C_23K VL without preamble" <223> "3C_23K VL without preamble

<220>

<223> 3C_23k VL without preamble "< 223 >" 3c_23k VL without preamble

<220>

<223> 3C_23K VL without leader sequence

<220>

<223> 3C_23K VL without leader sequence

<220>

<221> CDS

<222> 1..318

<400> 5

gac atc cag atg aca cag tcc cca tct acc ctg tct gct tcc gtg gga 48

gat cgg gtg act atc acc tgc aga gca agc tcc tcc gtg agg tac atc 96

gct tgg tac cag cag aag cca gga aag gcc cca aag ctg ctg acc tac 144

cca acc tcc tcc ctg aaa tcc ggg gtg ccc agc aga ttc tca ggc agt 192

ggc tcc ggc acc gaa ttc acc ctg acc atc agc tca ctg cag cct gac 240

gac ttc gca acc tac tac tgt ctg cag tgg agt agc tac cct tgg aca 288

ttc ggc ggc ggc acc aag gtg gag atc aag 318

<210> 6

<211> 106

<212> PRT

<213> Artificial sequence

<223> Synthetic construct

<223> "[ CDS ] 1..318" from SEQ ID NO 5"

<223> Synthetic construct

<223> Synthetic construct

<223> Synthetic construct

<220>

<223> Synthetic construct <223> synthetic

Construct <223> synthetic construct

[ CDS ] 1:318 from SEQ ID NO 5

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<400> 6

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 7

<211> 345

<212> DNA

<213> Artificial sequence

<223> "3C_23K VH without leader sequence";

<223> "3C_23K VH without leader sequence"

<223> "3C_23K VH without leader sequence"

<223> "3C_23K VH without leader sequence"

<220>

<223> 3C_23K VH without leader sequence "< 223 >" 3C_23K VH without leader sequence "< 223>

"3C_23K VH without leader sequence" <223> "3C_23K VH without leader sequence

<220>

<223> 3C_23K VH without leader sequence "< 223 >" 3C_23K VH without leader sequence

<220>

<223> 3C_23K VH without leader sequence

<220>

<223> 3C_23K VH without leader sequence

<220>

<221> CDS

<222> 1..345

<400> 7

cag gtg cgg ctg gtg cag agc ggg gcc gag gtg aag aag cct gga gcc 48

tca gtg aag gtg agt tgc aag gcc tcc ggt tac acc ttc acc agc tac 96

cac atc cac tgg gtc aga cag gct ccc ggc cag aga ctg gag tgg atg 144

ggc tgg atc tac cct gga gat gac tcc acc aag tac tcc cag aag ttc 192

cag ggt cgc gtg acc att acc agg gac acc agc gcc tcc act gcc tac 240

atg gag ctg tct tcc ctg aga tct gag gat acc gca gtc tac tac tgt 288

aca cgg ggg gac cgc ttt gct tac tgg ggg cag ggc act ctg gtg acc 336

gtc tcg agc 345

<210> 8

<211> 115

<212> PRT

<213> Artificial sequence

<223> Synthetic construct

<223> "[ CDS ] 1:345 from SEQ ID NO 7 ]"

<223> Synthetic construct

<223> Synthetic construct

<223> Synthetic construct

<220>

<223> Synthetic construct <223> synthetic

Construct <223> synthetic construct

[ CDS ] 1..345 from SEQ ID NO 7

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<400> 8

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 9

<211> 639

<212> DNA

<213> Artificial sequence

<223> "3C_23 light chain without leader".

<223> "3C_23 light chain without leader";

<223> "3C_23 light chain without leader sequence"

<223> "3C_23 light chain without leader sequence"

<220>

<223> Non-leader 3C_23 light chain "< 223 >" non-leader

3C_23 light chain "< 223 >" 3c_23 light chain without leader "< 223>

"3C_23 light chain without leader sequence

<220>

<223> Non-leader 3C_23 light chain "< 223 >" non-leader

3C_23 light chain

<220>

<223> 3C_23 light chain without leader sequence

<220>

<223> 3C_23 light chain without leader sequence

<220>

<221> CDS

<222> 1..639

<400> 9

gac atc cag atg aca cag tcc cca tct acc ctg tct gct tcc gtg gga 48

gat cgg gtg act atc acc tgc aga gca agc tcc tcc gtg agg tac atc 96

gct tgg tac cag cag aag cca gga aag gcc cca aag ctg ctg acc tac 144

cca acc tcc tcc ctg gaa tcc ggg gtg ccc agc aga ttc tca ggc agt 192

ggc tcc ggc acc gaa ttc acc ctg acc atc agc tca ctg cag cct gac 240

gac ttc gca acc tac tac tgt ctg cag tgg agt agc tac cct tgg aca 288

ttc ggc ggc ggc acc aag gtg gag atc aag cgg acc gtc gcc gca cca 336

agt gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga act 384

gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa 432

gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag 480

agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc agc 528

acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac gcc 576

tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc 624

aac agg gga gag tgt 639

<210> 10

<211> 213

<212> PRT

<213> Artificial sequence

<223> Synthetic construct

<223> "[ CDS ] 1..639" from SEQ ID NO 9"

<223> Synthetic construct

<223> Synthetic construct

<223> Synthetic construct

<220>

<223> Synthetic construct <223> synthetic

Construct <223> synthetic construct

[ CDS ] 1..639 from SEQ ID NO 9

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 11

<211> 1335

<212> DNA

<213> Artificial sequence

<223> "3C_23 heavy chain without leader".

<223> "3C_23 heavy chain without leader";

<223> "3C_23 heavy chain without leader sequence"

<223> "3C_23 heavy chain without leader sequence"

<220>

<223> Non-leader 3C_23 heavy chain "< 223 >" non-leader

3C_23 heavy chain "< 223 >" 3c_23 heavy chain without leader "< 223>

"3C_23 heavy chain without leader sequence

<220>

<223> Non-leader 3C_23 heavy chain "< 223 >" non-leader

3C_23 heavy chain

<220>

<223> 3C_23 heavy chain without leader sequence

<220>

<223> 3C_23 heavy chain without leader sequence

<220>

<221> CDS

<222> 1..1335

<400> 11

cag gtg cgg ctg gtg cag agc ggg gcc gag gtg aag aag cct gga gcc 48

tca gtg aag gtg agt tgc aag gcc tcc ggt tac acc ttc acc agc tac 96

cac atc cac tgg gtc aga cag gct ccc ggc cag aga ctg gag tgg atg 144

ggc tgg atc tac cct gga gat gac tcc acc aag tac tcc cag aag ttc 192

cag ggt cgc gtg acc att acc agg gac acc agc gcc tcc act gcc tac 240

atg gag ctg tct tcc ctg aga tct gag gat acc gca gtc tac tac tgt 288

aca cgg ggg gac cgc ttt gct tac tgg ggg cag ggc act ctg gtg acc 336

gtc tcg agc gcc agc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc 384

tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc 432

aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc 480

ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga 528

ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc 576

acc cag acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag 624

gtg gac aag aaa gtt gag ccc aaa tct tgt gac aaa act cac aca tgc 672

cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc 720

ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag 768

gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag 816

ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag 864

ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc 912

acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag 960

gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa 1008

gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc 1056

cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa 1104

ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag 1152

ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc 1200

tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag 1248

cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac 1296

cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa 1335

<210> 12

<211> 445

<212> PRT

<213> Artificial sequence

<223> Synthetic construct

<223> "[ CDS ] 1..1335 from SEQ ID NO 11 ]"

<223> Synthetic construct

<223> Synthetic construct

<223> Synthetic construct

<220>

<223> Synthetic construct <223> synthetic

Construct <223> synthetic construct

[ CDS ] 1..1335 from SEQ ID NO 11

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<400> 12

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 13

<211> 639

<212> DNA

<213> Artificial sequence

<223> "3C_23K light chain without leader";

<223> "3C_23K light chain without leader";

<223> "3C_23K light chain without leader sequence"

<223> "3C_23K light chain without leader sequence"

<220>

<223> Non-leader 3C_23K light chain "< 223 >" non-leader

3C_23K light chain "< 223 >" 3C_23K light chain without leader sequence "< 223>

"3C_23K light chain without leader sequence

<220>

<223> Non-leader 3C_23K light chain "< 223 >" non-leader

3C_23K light chain

<220>

<223> 3C_23K light chain without leader sequence

<220>

<223> 3C_23K light chain without leader sequence

<220>

<221> CDS

<222> 1..639

<400> 13

gac atc cag atg aca cag tcc cca tct acc ctg tct gct tcc gtg gga 48

gat cgg gtg act atc acc tgc aga gca agc tcc tcc gtg agg tac atc 96

gct tgg tac cag cag aag cca gga aag gcc cca aag ctg ctg acc tac 144

cca acc tcc tcc ctg aaa tcc ggg gtg ccc agc aga ttc tca ggc agt 192

ggc tcc ggc acc gaa ttc acc ctg acc atc agc tca ctg cag cct gac 240

gac ttc gca acc tac tac tgt ctg cag tgg agt agc tac cct tgg aca 288

ttc ggc ggc ggc acc aag gtg gag atc aag cgg acc gtc gcc gca cca 336

agt gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga act 384

gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa 432

gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag 480

agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc agc 528

acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac gcc 576

tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc 624

aac agg gga gag tgt 639

<210> 14

<211> 213

<212> PRT

<213> Artificial sequence

<223> Synthetic construct

<223> "[ CDS ] 1..639" from SEQ ID NO 13 "

<223> Synthetic construct

<223> Synthetic construct

<223> Synthetic construct

<220>

<223> Synthetic construct <223> synthetic

Construct <223> synthetic construct

[ CDS ] 1..639 from SEQ ID NO 13

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 15

<211> 1335

<212> DNA

<213> Artificial sequence

<223> "3C_23K heavy chain without leader";

<223> "3C_23K heavy chain without leader sequence"

<223> "3C_23K heavy chain without leader sequence"

<223> "3C_23K heavy chain without leader sequence"

<220>

<223> 3C_23k heavy chain without leader sequence "< 223 >" 3c_23k heavy chain without leader sequence

"< 223 >" 3C_23K heavy chain without leader sequence "< 223>

"3C_23K heavy chain without leader sequence

<220>

<223> 3C_23k heavy chain without leader sequence "< 223 >" 3c_23k heavy chain without leader sequence

<220>

<223> 3C_23K heavy chain without leader sequence

<220>

<223> 3C_23K heavy chain without leader sequence

<220>

<221> CDS

<222> 1..1335

<400> 15

cag gtg cgg ctg gtg cag agc ggg gcc gag gtg aag aag cct gga gcc 48

tca gtg aag gtg agt tgc aag gcc tcc ggt tac acc ttc acc agc tac 96

cac atc cac tgg gtc aga cag gct ccc ggc cag aga ctg gag tgg atg 144

ggc tgg atc tac cct gga gat gac tcc acc aag tac tcc cag aag ttc 192

cag ggt cgc gtg acc att acc agg gac acc agc gcc tcc act gcc tac 240

atg gag ctg tct tcc ctg aga tct gag gat acc gca gtc tac tac tgt 288

aca cgg ggg gac cgc ttt gct tac tgg ggg cag ggc act ctg gtg acc 336

gtc tcg agc gcc agc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc 384

tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc 432

aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc 480

ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga 528

ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc 576

acc cag acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag 624

gtg gac aag aaa gtt gag ccc aaa tct tgt gac aaa act cac aca tgc 672

cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc 720

ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag 768

gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag 816

ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag 864

ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc 912

acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag 960

gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa 1008

gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc 1056

cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa 1104

ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag 1152

ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc 1200

tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg cag 1248

cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac 1296

cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa 1335

<210> 16

<211> 445

<212> PRT

<213> Artificial sequence

<223> Synthetic construct

<223> "[ CDS ] 1..1335 from SEQ ID NO 15 ]"

<223> Synthetic construct

<223> Synthetic construct

<223> Synthetic construct

<220>

<223> Synthetic construct <223> synthetic

Construct <223> synthetic construct

[ CDS ] 1..1335 from SEQ ID NO 15

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<220>

<223> Synthetic construct

<400> 16

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 17

<211> 17

<212> PRT

<213> Chile person

<223> "Signal peptide" <223> Signal peptide

<223> Signal peptide "< 223> Signal peptide

<223> Signal peptide

<223> Signal peptide

<220>

<223> Signal peptide "< 223> Signal peptide <223>

Signal peptides

<220>

<223> Signal peptide "< 223> Signal peptide

<220>

<223> Signal peptide

<220>

<223> Signal peptide

<400> 17

Met Leu Gly Ser Leu Gly Leu Trp Ala Leu Leu Pro Thr Ala Val Glu

1 5 10 15

Ala

<210> 18

<211> 556

<212> PRT

<213> Chile person

<223> "Human AMHR-II lacking the signal peptide SEQ ID NO: 17" <223> human AMHR-II <223> lacking the signal peptide SEQ ID NO: 17 human AMHR-II

<223> Human AMHR-II lacking the signal peptide SEQ ID NO: 17 "< 223> human AMHR-II lacking the signal peptide SEQ ID NO: 17

<223> Human AMHR-II lacking the Signal peptide SEQ ID NO: 17

<223> Human AMHR-II lacking the Signal peptide SEQ ID NO: 17

<220>

<223> Human AMHR-II "< 223> lacking the Signal peptide SEQ ID NO: 17

Human AMHR-II <223> lacking the signal peptide SEQ ID NO 17

Human AMHR-II <223> lacking the signal peptide SEQ ID NO 17

Human AMHR-II lacking the signal peptide SEQ ID NO 17

<220>

<223> Human AMHR-II "< 223> lacking the Signal peptide SEQ ID NO: 17

Human AMHR-II lacking the signal peptide SEQ ID NO 17

<220>

<223> Human AMHR-II lacking the Signal peptide SEQ ID NO: 17

<220>

<223> Human AMHR-II lacking the Signal peptide SEQ ID NO: 17

<400> 18

Pro Pro Asn Arg Arg Thr Cys Val Phe Phe Glu Ala Pro Gly Val Arg

1 5 10 15

Gly Ser Thr Lys Thr Leu Gly Glu Leu Leu Asp Thr Gly Thr Glu Leu

20 25 30

Pro Arg Ala Ile Arg Cys Leu Tyr Ser Arg Cys Cys Phe Gly Ile Trp

35 40 45

Asn Leu Thr Gln Asp Arg Ala Gln Val Glu Met Gln Gly Cys Arg Asp

50 55 60

Ser Asp Glu Pro Gly Cys Glu Ser Leu His Cys Asp Pro Ser Pro Arg

65 70 75 80

Ala His Pro Ser Pro Gly Ser Thr Leu Phe Thr Cys Ser Cys Gly Thr

85 90 95

Asp Phe Cys Asn Ala Asn Tyr Ser His Leu Pro Pro Pro Gly Ser Pro

100 105 110

Gly Thr Pro Gly Ser Gln Gly Pro Gln Ala Ala Pro Gly Glu Ser Ile

115 120 125

Trp Met Ala Leu Val Leu Leu Gly Leu Phe Leu Leu Leu Leu Leu Leu

130 135 140

Leu Gly Ser Ile Ile Leu Ala Leu Leu Gln Arg Lys Asn Tyr Arg Val

145 150 155 160

Arg Gly Glu Pro Val Pro Glu Pro Arg Pro Asp Ser Gly Arg Asp Trp

165 170 175

Ser Val Glu Leu Gln Glu Leu Pro Glu Leu Cys Phe Ser Gln Val Ile

180 185 190

Arg Glu Gly Gly His Ala Val Val Trp Ala Gly Gln Leu Gln Gly Lys

195 200 205

Leu Val Ala Ile Lys Ala Phe Pro Pro Arg Ser Val Ala Gln Phe Gln

210 215 220

Ala Glu Arg Ala Leu Tyr Glu Leu Pro Gly Leu Gln His Asp His Ile

225 230 235 240

Val Arg Phe Ile Thr Ala Ser Arg Gly Gly Pro Gly Arg Leu Leu Ser

245 250 255

Gly Pro Leu Leu Val Leu Glu Leu His Pro Lys Gly Ser Leu Cys His

260 265 270

Tyr Leu Thr Gln Tyr Thr Ser Asp Trp Gly Ser Ser Leu Arg Met Ala

275 280 285

Leu Ser Leu Ala Gln Gly Leu Ala Phe Leu His Glu Glu Arg Trp Gln

290 295 300

Asn Gly Gln Tyr Lys Pro Gly Ile Ala His Arg Asp Leu Ser Ser Gln

305 310 315 320

Asn Val Leu Ile Arg Glu Asp Gly Ser Cys Ala Ile Gly Asp Leu Gly

325 330 335

Leu Ala Leu Val Leu Pro Gly Leu Thr Gln Pro Pro Ala Trp Thr Pro

340 345 350

Thr Gln Pro Gln Gly Pro Ala Ala Ile Met Glu Ala Gly Thr Gln Arg

355 360 365

Tyr Met Ala Pro Glu Leu Leu Asp Lys Thr Leu Asp Leu Gln Asp Trp

370 375 380

Gly Met Ala Leu Arg Arg Ala Asp Ile Tyr Ser Leu Ala Leu Leu Leu

385 390 395 400

Trp Glu Ile Leu Ser Arg Cys Pro Asp Leu Arg Pro Asp Ser Ser Pro

405 410 415

Pro Pro Phe Gln Leu Ala Tyr Glu Ala Glu Leu Gly Asn Thr Pro Thr

420 425 430

Ser Asp Glu Leu Trp Ala Leu Ala Val Gln Glu Arg Arg Arg Pro Tyr

435 440 445

Ile Pro Ser Thr Trp Arg Cys Phe Ala Thr Asp Pro Asp Gly Leu Arg

450 455 460

Glu Leu Leu Glu Asp Cys Trp Asp Ala Asp Pro Glu Ala Arg Leu Thr

465 470 475 480

Ala Glu Cys Val Gln Gln Arg Leu Ala Ala Leu Ala His Pro Gln Glu

485 490 495

Ser His Pro Phe Pro Glu Ser Cys Pro Arg Gly Cys Pro Pro Leu Cys

500 505 510

Pro Glu Asp Cys Thr Ser Ile Pro Ala Pro Thr Ile Leu Pro Cys Arg

515 520 525

Pro Gln Arg Ser Ala Cys His Phe Ser Val Gln Gln Gly Pro Cys Ser

530 535 540

Arg Asn Pro Gln Pro Ala Cys Thr Leu Ser Pro Val

545 550 555

<210> 19

<211> 115

<212> PRT

<213> Artificial sequence

<223> 3C23K/3C23 <223> 3C23K/3C23 <223> 3C23K/3C23 <223> 3C23K/3C23

<223> 3C23K/3C23 <223> 3C23K/3C23

<223> 3C23K/3C23

<223> 3C23K/3C23

<220>

<223> 3C23K/3C23 <223> 3C23K/3C23 <223> 3C23K/3C23 <223> 3C23K/3C23

<220>

<223> 3C23K/3C23 <223> 3C23K/3C23

<220>

<223> 3C23K/3C23

<220>

<223> 3C23K/3C23

<400> 19

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 20

<211> 115

<212> PRT

<213> Artificial sequence

<223> 3C23KR/6B78 <223> 3C23KR/6B78 <223> 3C23KR/6B78 <223> 3C23KR/6B78

<223> 3C23KR/6B78 <223> 3C23KR/6B78

<223> 3C23KR/6B78

<223> 3C23KR/6B78

<220>

<223> 3C23KR/6B78 <223> 3C23KR/6B78 <223> 3C23KR/6B78 <223> 3C23KR/6B78

<220>

<223> 3C23KR/6B78 <223> 3C23KR/6B78

<220>

<223> 3C23KR/6B78

<220>

<223> 3C23KR/6B78

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 21

<211> 115

<212> PRT

<213> Artificial sequence

<223> 5B42 <223> 5B42 <223> 5B42 <223> 5B42

<223> 5B42 <223> 5B42

<223> 5B42

<223> 5B42

<220>

<223> 5B42 <223> 5B42 <223> 5B42 <223> 5B42

<220>

<223> 5B42 <223> 5B42

<220>

<223> 5B42

<220>

<223> 5B42

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Ala Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 22

<211> 115

<212> PRT

<213> Artificial sequence

<223> K4D-24/6C59 <223> K4D-24/6C59 <223> K4D-24/6C59 <223> K4D-24/6C59

<223> K4D-24/6C59 <223> K4D-24/6C59

<223> K4D-24/6C59

<223> K4D-24/6C59

<220>

<223> K4D-24/6C59 <223> K4D-24/6C59 <223> K4D-24/6C59 <223> K4D-24/6C59

<220>

<223> K4D-24/6C59 <223> K4D-24/6C59

<220>

<223> K4D-24/6C59

<220>

<223> K4D-24/6C59

<400> 22

Arg Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 23

<211> 115

<212> PRT

<213> Artificial sequence

<223> K4D-20 <223> K4D-20 <223> K4D-20 <223> K4D-20

<223> K4D-20 <223> K4D-20

<223> K4D-20

<223> K4D-20

<220>

<223> K4D-20 <223> K4D-20 <223> K4D-20 <223> K4D-20

<220>

<223> K4D-20 <223> K4D-20

<220>

<223> K4D-20

<220>

<223> K4D-20

<400> 23

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Asn

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 24

<211> 115

<212> PRT

<213> Artificial sequence

<223> K4A-12 <223> K4A-12 <223> K4A-12 <223> K4A-12

<223> K4A-12 <223> K4A-12

<223> K4A-12

<223> K4A-12

<220>

<223> K4A-12 <223> K4A-12 <223> K4A-12 <223> K4A-12

<220>

<223> K4A-12 <223> K4A-12

<220>

<223> K4A-12

<220>

<223> K4A-12

<400> 24

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 25

<211> 115

<212> PRT

<213> Artificial sequence

<223> K5D05 <223> K5D05 <223> K5D05 <223> K5D05

<223> K5D05 <223> K5D05

<223> K5D05

<223> K5D05

<220>

<223> K5D05 <223> K5D05 <223> K5D05 <223> K5D05

<220>

<223> K5D05 <223> K5D05

<220>

<223> K5D05

<220>

<223> K5D05

<400> 25

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 26

<211> 115

<212> PRT

<213> Artificial sequence

<223> K5D-14 <223> K5D-14 <223> K5D-14 <223> K5D-14

<223> K5D-14 <223> K5D-14

<223> K5D-14

<223> K5D-14

<220>

<223> K5D-14 <223> K5D-14 <223> K5D-14 <223> K5D-14

<220>

<223> K5D-14 <223> K5D-14

<220>

<223> K5D-14

<220>

<223> K5D-14

<400> 26

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 27

<211> 115

<212> PRT

<213> Artificial sequence

<223> K4D-123 <223> K4D-123 <223> K4D-123 <223> K4D-123

<223> K4D-123 <223> K4D-123

<223> K4D-123

<223> K4D-123

<220>

<223> K4D-123 <223> K4D-123 <223> K4D-123 <223> K4D-123

<220>

<223> K4D-123 <223> K4D-123

<220>

<223> K4D-123

<220>

<223> K4D-123

<400> 27

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 28

<211> 115

<212> PRT

<213> Artificial sequence

<223> K4D-127/6C07 <223> K4D-127/6C07 <223> K4D-127/6C07 <223> K4D-127/6C07

<223> K4D-127/6C07 <223> K4D-127/6C07

<223> K4D-127/6C07

<223> K4D-127/6C07

<220>

<223> K4D-127/6C07 <223> K4D-127/6C07 <223> K4D-127/6C07 <223>

K4D-127/6C07

<220>

<223> K4D-127/6C07 <223> K4D-127/6C07

<220>

<223> K4D-127/6C07

<220>

<223> K4D-127/6C07

<400> 28

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Thr Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 29

<211> 115

<212> PRT

<213> Artificial sequence

<223> 5C14 <223> 5C14 <223> 5C14 <223> 5C14

<223> 5C14 <223> 5C14

<223> 5C14

<223> 5C14

<220>

<223> 5C14 <223> 5C14 <223> 5C14 <223> 5C14

<220>

<223> 5C14 <223> 5C14

<220>

<223> 5C14

<220>

<223> 5C14

<400> 29

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Phe Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 30

<211> 115

<212> PRT

<213> Artificial sequence

<223> 5C26 <223> 5C26 <223> 5C26 <223> 5C26

<223> 5C26 <223> 5C26

<223> 5C26

<223> 5C26

<220>

<223> 5C26 <223> 5C26 <223> 5C26 <223> 5C26

<220>

<223> 5C26 <223> 5C26

<220>

<223> 5C26

<220>

<223> 5C26

<400> 30

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Met Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 31

<211> 115

<212> PRT

<213> Artificial sequence

<223> 5C27 <223> 5C27 <223> 5C27 <223> 5C27

<223> 5C27 <223> 5C27

<223> 5C27

<223> 5C27

<220>

<223> 5C27 <223> 5C27 <223> 5C27 <223> 5C27

<220>

<223> 5C27 <223> 5C27

<220>

<223> 5C27

<220>

<223> 5C27

<400> 31

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Pro Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 32

<211> 115

<212> PRT

<213> Artificial sequence

<223> 5C60 <223> 5C60 <223> 5C60 <223> 5C60

<223> 5C60 <223> 5C60

<223> 5C60

<223> 5C60

<220>

<223> 5C60 <223> 5C60 <223> 5C60 <223> 5C60

<220>

<223> 5C60 <223> 5C60

<220>

<223> 5C60

<220>

<223> 5C60

<400> 32

Gln Val Arg Leu Val Gln Ser Gly Ala Lys Val Arg Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 33

<211> 115

<212> PRT

<213> Artificial sequence

<223> 6C13 <223> 6C13 <223> 6C13 <223> 6C13

<223> 6C13 <223> 6C13

<223> 6C13

<223> 6C13

<220>

<223> 6C13 <223> 6C13 <223> 6C13 <223> 6C13

<220>

<223> 6C13 <223> 6C13

<220>

<223> 6C13

<220>

<223> 6C13

<400> 33

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Glu Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 34

<211> 115

<212> PRT

<213> Artificial sequence

<223> 6C18 <223> 6C18 <223> 6C18 <223> 6C18

<223> 6C18 <223> 6C18

<223> 6C18

<223> 6C18

<220>

<223> 6C18 <223> 6C18 <223> 6C18 <223> 6C18

<220>

<223> 6C18 <223> 6C18

<220>

<223> 6C18

<220>

<223> 6C18

<400> 34

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 35

<211> 115

<212> PRT

<213> Artificial sequence

<223> 6C54 <223> 6C54 <223> 6C54 <223> 6C54

<223> 6C54 <223> 6C54

<223> 6C54

<223> 6C54

<220>

<223> 6C54 <223> 6C54 <223> 6C54 <223> 6C54

<220>

<223> 6C54 <223> 6C54

<220>

<223> 6C54

<220>

<223> 6C54

<400> 35

Gln Val Arg Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

His Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Arg Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 36

<211> 106

<212> PRT

<213> Artificial sequence

<223> 3C23K <223> 3C23K <223> 3C23K <223> 3C23K

<223> 3C23K <223> 3C23K

<223> 3C23K

<223> 3C23K

<220>

<223> 3C23K <223> 3C23K <223> 3C23K <223> 3C23K

<220>

<223> 3C23K <223> 3C23K

<220>

<223> 3C23K

<220>

<223> 3C23K

<400> 36

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 37

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-K55E <223> L-K55E <223> L-K55E <223> L-K55E

<223> L-K55E <223> L-K55E

<223> L-K55E

<223> L-K55E

<220>

<223> L-K55E <223> L-K55E <223> L-K55E <223> L-K55E

<220>

<223> L-K55E <223> L-K55E

<220>

<223> L-K55E

<220>

<223> L-K55E

<400> 37

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 38

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-T48I, L-P50S <223> L-T48I, L-P50S <223> L-T48I, L-P50S <223> L-T48I, L-P50S

<223> L-T48I, L-P50S <223> L-T48I, L-P50S

<223> L-T48I, L-P50S

<223> L-T48I, L-P50S

<220>

<223> L-T48I, L-P50S <223> L-T48I, L-P50S <223> L-T48I, L-P50S <223>

L-T48I, L-P50S

<220>

<223> L-T48I, L-P50S <223> L-T48I, L-P50S

<220>

<223> L-T48I, L-P50S

<220>

<223> L-T48I, L-P50S

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 39

<211> 106

<212> PRT

<213> Artificial sequence

<223> LT48I, L-K55E <223> LT48I, L-K55E <223> LT48I, L-K55E <223> LT48I, L-K55E

<223> LT48I, L-K55E <223> LT48I, L-K55E

<223> LT48I, L-K55E

<223> LT48I, L-K55E

<220>

<223> LT48I, L-K55E <223> LT48I, L-K55E <223> LT48I, L-K55E <223>

LT48I, L-K55E

<220>

<223> LT48I, L-K55E <223> LT48I, L-K55E

<220>

<223> LT48I, L-K55E

<220>

<223> LT48I, L-K55E

<400> 39

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Pro Thr Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 40

<211> 106

<212> PRT

<213> Artificial sequence

<223> LS27P, L-S28P <223> LS27P, L-S28P <223> LS27P, L-S28P <223> LS27P, L-S28P

<223> LS27P, L-S28P <223> LS27P, L-S28P

<223> LS27P, L-S28P

<223> LS27P, L-S28P

<220>

<223> LS27P, L-S28P <223> LS27P, L-S28P <223> LS27P, L-S28P <223>

LS27P, L-S28P

<220>

<223> LS27P, L-S28P <223> LS27P, L-S28P

<220>

<223> LS27P, L-S28P

<220>

<223> LS27P, L-S28P

<400> 40

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Pro Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 41

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-M4L, L-T20A <223> L-M4L, L-T20A <223> L-M4L, L-T20A <223> L-M4L, L-T20A

<223> L-M4L, L-T20A <223> L-M4L, L-T20A

<223> L-M4L, L-T20A

<223> L-M4L, L-T20A

<220>

<223> L-M4L, L-T20A <223> L-M4L, L-T20A <223> L-M4L, L-T20A <223>

L-M4L, L-T20A

<220>

<223> L-M4L, L-T20A <223> L-M4L, L-T20A

<220>

<223> L-M4L, L-T20A

<220>

<223> L-M4L, L-T20A

<400> 41

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ala Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 42

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-S27P <223> L-S27P <223> L-S27P <223> L-S27P

<223> L-S27P <223> L-S27P

<223> L-S27P

<223> L-S27P

<220>

<223> L-S27P <223> L-S27P <223> L-S27P <223> L-S27P

<220>

<223> L-S27P <223> L-S27P

<220>

<223> L-S27P

<220>

<223> L-S27P

<400> 42

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Pro Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 43

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-M4L, L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W

<223> L-M4L, L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W

<223> L-M4L, L-S9P, L-R31W

<223> L-M4L, L-S9P, L-R31W

<220>

<223> L-M4L, L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W <223> L-M4L,

L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W

<220>

<223> L-M4L, L-S9P, L-R31W <223> L-M4L, L-S9P, L-R31W

<220>

<223> L-M4L, L-S9P, L-R31W

<220>

<223> L-M4L, L-S9P, L-R31W

<400> 43

Asp Ile Gln Leu Thr Gln Ser Pro Pro Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Trp Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 44

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-M4L <223> L-M4L <223> L-M4L <223> L-M4L

<223> L-M4L <223> L-M4L

<223> L-M4L

<223> L-M4L

<220>

<223> L-M4L <223> L-M4L <223> L-M4L <223> L-M4L

<220>

<223> L-M4L <223> L-M4L

<220>

<223> L-M4L

<220>

<223> L-M4L

<400> 44

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 45

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-I33T <223> L-I33T <223> L-I33T <223> L-I33T

<223> L-I33T <223> L-I33T

<223> L-I33T

<223> L-I33T

<220>

<223> L-I33T <223> L-I33T <223> L-I33T <223> L-I33T

<220>

<223> L-I33T <223> L-I33T

<220>

<223> L-I33T

<220>

<223> L-I33T

<400> 45

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Thr

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 46

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-M4L, L-K39E <223> L-M4L, L-K39E <223> L-M4L, L-K39E <223> L-M4L, L-K39E

<223> L-M4L, L-K39E <223> L-M4L, L-K39E

<223> L-M4L, L-K39E

<223> L-M4L, L-K39E

<220>

<223> L-M4L, L-K39E <223> L-M4L, L-K39E <223> L-M4L, L-K39E <223>

L-M4L, L-K39E

<220>

<223> L-M4L, L-K39E <223> L-M4L, L-K39E

<220>

<223> L-M4L, L-K39E

<220>

<223> L-M4L, L-K39E

<400> 46

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Glu Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 47

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-T22P <223> L-T22P <223> L-T22P <223> L-T22P

<223> L-T22P <223> L-T22P

<223> L-T22P

<223> L-T22P

<220>

<223> L-T22P <223> L-T22P <223> L-T22P <223> L-T22P

<220>

<223> L-T22P <223> L-T22P

<220>

<223> L-T22P

<220>

<223> L-T22P

<400> 47

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Pro Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 48

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-Y32D <223> L-Y32D <223> L-Y32D <223> L-Y32D

<223> L-Y32D <223> L-Y32D

<223> L-Y32D

<223> L-Y32D

<220>

<223> L-Y32D <223> L-Y32D <223> L-Y32D <223> L-Y32D

<220>

<223> L-Y32D <223> L-Y32D

<220>

<223> L-Y32D

<220>

<223> L-Y32D

<400> 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Asp Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 49

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-Q37H <223> L-Q37H <223> L-Q37H <223> L-Q37H

<223> L-Q37H <223> L-Q37H

<223> L-Q37H

<223> L-Q37H

<220>

<223> L-Q37H <223> L-Q37H <223> L-Q37H <223> L-Q37H

<220>

<223> L-Q37H <223> L-Q37H

<220>

<223> L-Q37H

<220>

<223> L-Q37H

<400> 49

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr His Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 50

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-G97S <223> L-G97S <223> L-G97S <223> L-G97S

<223> L-G97S <223> L-G97S

<223> L-G97S

<223> L-G97S

<220>

<223> L-G97S <223> L-G97S <223> L-G97S <223> L-G97S

<220>

<223> L-G97S <223> L-G97S

<220>

<223> L-G97S

<220>

<223> L-G97S

<400> 50

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Ser Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 51

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-S12P <223> L-S12P <223> L-S12P <223> L-S12P

<223> L-S12P <223> L-S12P

<223> L-S12P

<223> L-S12P

<220>

<223> L-S12P <223> L-S12P <223> L-S12P <223> L-S12P

<220>

<223> L-S12P <223> L-S12P

<220>

<223> L-S12P

<220>

<223> L-S12P

<400> 51

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Pro Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 52

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-19A <223> L-19A <223> L-19A <223> L-19A

<223> L-19A <223> L-19A

<223> L-19A

<223> L-19A

<220>

<223> L-19A <223> L-19A <223> L-19A <223> L-19A

<220>

<223> L-19A <223> L-19A

<220>

<223> L-19A

<220>

<223> L-19A

<400> 52

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Ala Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 53

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-T72A <223> L-T72A <223> L-T72A <223> L-T72A

<223> L-T72A <223> L-T72A

<223> L-T72A

<223> L-T72A

<220>

<223> L-T72A <223> L-T72A <223> L-T72A <223> L-T72A

<220>

<223> L-T72A <223> L-T72A

<220>

<223> L-T72A

<220>

<223> L-T72A

<400> 53

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Ala Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 54

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-R31W <223> L-R31W <223> L-R31W <223> L-R31W

<223> L-R31W <223> L-R31W

<223> L-R31W

<223> L-R31W

<220>

<223> L-R31W <223> L-R31W <223> L-R31W <223> L-R31W

<220>

<223> L-R31W <223> L-R31W

<220>

<223> L-R31W

<220>

<223> L-R31W

<400> 54

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Trp Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 55

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-M4L, L-M39K <223> L-M4L, L-M39K <223> L-M4L, L-M39K <223> L-M4L, L-M39K

<223> L-M4L, L-M39K <223> L-M4L, L-M39K

<223> L-M4L, L-M39K

<223> L-M4L, L-M39K

<220>

<223> L-M4L, L-M39K <223> L-M4L, L-M39K <223> L-M4L, L-M39K <223>

L-M4L, L-M39K

<220>

<223> L-M4L, L-M39K <223> L-M4L, L-M39K

<220>

<223> L-M4L, L-M39K

<220>

<223> L-M4L, L-M39K

<400> 55

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Met Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 56

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-I2N <223> L-I2N <223> L-I2N <223> L-I2N

<223> L-I2N <223> L-I2N

<223> L-I2N

<223> L-I2N

<220>

<223> L-I2N <223> L-I2N <223> L-I2N <223> L-I2N

<220>

<223> L-I2N <223> L-I2N

<220>

<223> L-I2N

<220>

<223> L-I2N

<400> 56

Asp Asn Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 57

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-G63C, L-W91C <223> L-G63C, L-W91C <223> L-G63C, L-W91C <223> L-G63C, L-W91C

<223> L-G63C, L-W91C <223> L-G63C, L-W91C

<223> L-G63C, L-W91C

<223> L-G63C, L-W91C

<220>

<223> L-G63C, L-W91C <223> L-G63C, L-W91C <223> L-G63C, L-W91C <223>

L-G63C, L-W91C

<220>

<223> L-G63C, L-W91C <223> L-G63C, L-W91C

<220>

<223> L-G63C, L-W91C

<220>

<223> L-G63C, L-W91C

<400> 57

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Cys Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Cys Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 58

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-R31G <223> L-R31G <223> L-R31G <223> L-R31G

<223> L-R31G <223> L-R31G

<223> L-R31G

<223> L-R31G

<220>

<223> L-R31G <223> L-R31G <223> L-R31G <223> L-R31G

<220>

<223> L-R31G <223> L-R31G

<220>

<223> L-R31G

<220>

<223> L-R31G

<400> 58

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Gly Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 59

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-I75F <223> L-I75F <223> L-I75F <223> L-I75F

<223> L-I75F <223> L-I75F

<223> L-I75F

<223> L-I75F

<220>

<223> L-I75F <223> L-I75F <223> L-I75F <223> L-I75F

<220>

<223> L-I75F <223> L-I75F

<220>

<223> L-I75F

<220>

<223> L-I75F

<400> 59

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Phe Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 60

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-I2T <223> L-I2T <223> L-I2T <223> L-I2T

<223> L-I2T <223> L-I2T

<223> L-I2T

<223> L-I2T

<220>

<223> L-I2T <223> L-I2T <223> L-I2T <223> L-I2T

<220>

<223> L-I2T <223> L-I2T

<220>

<223> L-I2T

<220>

<223> L-I2T

<400> 60

Asp Thr Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 61

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-I2T, L-K42R <223> L-I2T, L-K42R <223> L-I2T, L-K42R <223> L-I2T, L-K42R

<223> L-I2T, L-K42R <223> L-I2T, L-K42R

<223> L-I2T, L-K42R

<223> L-I2T, L-K42R

<220>

<223> L-I2T, L-K42R <223> L-I2T, L-K42R <223> L-I2T, L-K42R <223>

L-I2T, L-K42R

<220>

<223> L-I2T, L-K42R <223> L-I2T, L-K42R

<220>

<223> L-I2T, L-K42R

<220>

<223> L-I2T, L-K42R

<400> 61

Asp Thr Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 62

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-Y49H <223> L-Y49H <223> L-Y49H <223> L-Y49H

<223> L-Y49H <223> L-Y49H

<223> L-Y49H

<223> L-Y49H

<220>

<223> L-Y49H <223> L-Y49H <223> L-Y49H <223> L-Y49H

<220>

<223> L-Y49H <223> L-Y49H

<220>

<223> L-Y49H

<220>

<223> L-Y49H

<400> 62

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr His

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 63

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-M4L, L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E

<223> L-M4L, L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E

<223> L-M4L, L-T20S, L-K39E

<223> L-M4L, L-T20S, L-K39E

<220>

<223> L-M4L, L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E <223> L-M4L,

L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E

<220>

<223> L-M4L, L-T20S, L-K39E <223> L-M4L, L-T20S, L-K39E

<220>

<223> L-M4L, L-T20S, L-K39E

<220>

<223> L-M4L, L-T20S, L-K39E

<400> 63

Asp Ile Gln Leu Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Glu Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 64

<211> 106

<212> PRT

<213> Artificial sequence

<223> L-T69P <223> L-T69P <223> L-T69P <223> L-T69P

<223> L-T69P <223> L-T69P

<223> L-T69P

<223> L-T69P

<220>

<223> L-T69P <223> L-T69P <223> L-T69P <223> L-T69P

<220>

<223> L-T69P <223> L-T69P

<220>

<223> L-T69P

<220>

<223> L-T69P

<400> 64

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Arg Tyr Ile

20 25 30

Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Thr Tyr

35 40 45

Pro Thr Ser Ser Leu Lys Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Pro Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Trp Ser Ser Tyr Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 65

<211> 10

<212> PRT

<213> Artificial sequence

<223> "CDRL-1 of anti-AMHRII antibody"

<223> CDRL-1 of anti-AMHRII antibody

<220>

<223> CDRL-1 of anti-AMHRII antibody

<220>

<223> CDRL-1 of anti-AMHRII antibody

<220>

<221> Variant

<222> 4

<223> Xaa at position 4 is S or P

<220>

<221> Variant

<222> 5

<223> Xaa at position 5 is S or P

<220>

<221> Variant

<222> 7

<223> Xaa at position 7 is R or W or G

<220>

<221> Variant

<222> 8

<223> Xaa at position 8 is T or D

<220>

<221> Variant

<222> 9

<223> Xaa at position 9 is I or T

<400> 65

Arg Ala Ser Xaa Xaa Val Xaa Xaa Xaa Ala

1 5 10

<210> 66

<211> 7

<212> PRT

<213> Artificial sequence

<223> "CDRL-2 of anti-AMHRII antibody"

<223> CDRL-2 of anti-AMHRII antibody

<220>

<223> CDRL-2 of anti-AMHRII antibody

<220>

<223> CDRL-2 of anti-AMHRII antibody

<220>

<221> Variant

<222> 6

<223> Xaa at position 6 is K or E

<400> 66

Pro Thr Ser Ser Leu Xaa Ser

1 5

<210> 67

<211> 9

<212> PRT

<213> Artificial sequence

<223> "CDRL-3 of anti-AMHRII antibody"

<223> CDRL-3 of anti-AMHRII antibody

<220>

<223> CDRL-3 of anti-AMHRII antibody

<220>

<223> CDRL-3 of anti-AMHRII antibody

<400> 67

Leu Gln Trp Ser Ser Tyr Pro Trp Thr

1 5

<210> 68

<211> 13

<212> PRT

<213> Artificial sequence

<223> "CDRH-1 of anti-AMHRII antibody"

<223> CDRH-1 of anti-AMHRII antibody

<220>

<223> CDRH-1 of anti-AMHRII antibody

<220>

<223> CDRH-1 of anti-AMHRII antibody

<220>

<221> Variant

<222> 6

<223> Xaa at position 6 is S or T

<220>

<221> Variant

<222> 9

<223> Xaa at position 9 is S or G

<220>

<221> Variant

<222> 10

<223> Xaa at position 10 is Y or N

<400> 68

Lys Ala Ser Gly Tyr Xaa Phe Thr Xaa Xaa His Ile His

1 5 10

<210> 69

<211> 17

<212> PRT

<213> Artificial sequence

<223> "CDRH-2 of anti-AMHRII antibody"

<223> CDRH-2 of anti-AMHRII antibody

<220>

<223> CDRH-2 of anti-AMHRII antibody

<220>

<223> CDRH-2 of anti-AMHRII antibody

<220>

<221> Variant

<222> 5

<223> Xaa at position 5 is G or E

<400> 69

Trp Ile Tyr Pro Xaa Asp Asp Ser Thr Lys Tyr Ser Gln Lys Phe Gln

1 5 10 15

Gly

<210> 70

<211> 6

<212> PRT

<213> Artificial sequence

<223> "CDRH-3 of anti-AMHRII antibody"

<223> CDRH-3 of anti-AMHRII antibody

<220>

<223> CDRH-3 of anti-AMHRII antibody

<220>

<223> CDRH-3 of anti-AMHRII antibody

<400> 70

Gly Asp Arg Phe Ala Tyr

1 5

<210> 71

<211> 20

<212> DNA

<213> Artificial sequence

<223> "Forward primer of AMHR 2"

<223> "Forward primer of AMHR 2"

<220>

<223> Forward primer for AMHR2

<220>

<223> Forward primer for AMHR2

<400> 71

tctggatggc actggtgctg 20

<210> 72

<211> 20

<212> DNA

<213> Artificial sequence

<223> "Reverse primer for AMHR 2"

<223> "Reverse primer for AMHR 2"

<220>

<223> Reverse primer for AMHR2

<220>

<223> Reverse primer for AMHR2

<400> 72

agcagggcca agatgatgct 20

<210> 73

<211> 21

<212> DNA

<213> Artificial sequence

<223> "Forward primer of TBP"

<223> "Forward primer of TBP"

<220>

<223> Forward primer of TBP

<220>

<223> Forward primer of TBP

<400> 73

tgcacaggag ccaagagtga a 21

<210> 74

<211> 20

<212> DNA

<213> Artificial sequence

<223> "Reverse primer of TBP"

<223> "Reverse primer of TBP"

<220>

<223> Reverse primer of TBP

<220>

<223> Reverse primer of TBP

<400> 74

cacatcacag ctccccacca 20

Claims (6)

1. Use of a monoclonal antibody in the manufacture of a medicament for preventing or treating lung cancer in a patient having lung cancer selected from the group consisting of: epidermoid non-small cell lung cancer, adenocarcinoma non-small cell lung cancer, large cell non-small cell lung cancer, squamous cell carcinoma non-small cell lung cancer, polymorphic cell carcinoma non-small cell lung cancer, and neuroendocrine non-small cell lung cancer, wherein the cancer cells of the lung cancer express human AMHRII on their membranes,

Wherein the monoclonal antibody comprises a VL and a VH, wherein: VL comprises CDR1, CDR2 and CDR3, said CDR1, CDR2 and CDR3 consisting of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in VL of the amino acid sequence of SEQ ID NO: 6; and, the VH comprises CDR1, CDR2 and CDR3, said CDR1, CDR2 and CDR3 consisting of the amino acid sequences of CDR1, CDR2 and CDR3 shown in the VH of the amino acid sequence of SEQ ID NO: 8; or alternatively

Wherein the monoclonal antibody comprises a VL and a VH, wherein: VL comprises CDR1, CDR2 and CDR3, said CDR1, CDR2 and CDR3 consisting of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in VL of the amino acid sequence of SEQ ID NO: 2; and, the VH comprises CDR1, CDR2 and CDR3, said CDR1, CDR2 and CDR3 consisting of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in the VH of the amino acid sequence of SEQ ID NO: 4.

2. The use of claim 1, wherein the monoclonal antibody is selected from the group consisting of:

a) A monoclonal antibody comprising a light chain variable region ("VL") of SEQ ID No.2 and a heavy chain variable region ("VH") of SEQ ID No. 4;

b) A monoclonal antibody comprising a VL of SEQ ID No.6 and a VH of SEQ ID No. 8;

c) A monoclonal antibody comprising a light chain of SEQ ID No.10 and a heavy chain of SEQ ID No. 12; and

D) A monoclonal antibody comprising a light chain of SEQ ID No. 14 and a heavy chain of SEQ ID No. 16.

3. The use of claim 1, wherein the monoclonal antibody comprises CDRs consisting of the sequences:

- CDRL-1：RASSVRYIA；

- CDRL-2：PTSSLKS；

- CDRL-3：LQWSSYPWT；

-CDRH-1：KASGYTFTSYHIH；

CDRH-2: WIYPGDDSTKYSQKFQG; and

- CDRH-3：GDRFAY。

4. The use of claim 1, wherein the monoclonal antibody comprises CDRs consisting of the sequences:

- CDRL-1：RASSVRYIA；

- CDRL-2：PTSSLES；

- CDRL-3：LQWSSYPWT；

-CDRH-1：KASGYTFTSYHIH；

CDRH-2: WIYPGDDSTKYSQKFQG; and

- CDRH-3：GDRFAY。

5. The use of any one of claims 1 to 4, wherein the monoclonal antibody is combined with one or more different anticancer agents.

6. The use of a monoclonal antibody in the preparation of a kit for determining whether an individual having lung cancer is likely to respond to cancer treatment with the monoclonal antibody,

Wherein the lung cancer is selected from the group consisting of: epidermoid non-small cell lung cancer, adenocarcinoma non-small cell lung cancer, large cell non-small cell lung cancer, squamous cell carcinoma non-small cell lung cancer, polymorphous cell carcinoma non-small cell lung cancer and neuroendocrine non-small cell lung cancer; wherein the cancer cells of the lung cancer express human AMHRII on their membranes, and

Wherein the monoclonal antibody comprises a VL and a VH, wherein: VL comprises CDR1, CDR2 and CDR3, said CDR1, CDR2 and CDR3 consisting of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in VL of the amino acid sequence of SEQ ID NO: 6; and, the VH comprises CDR1, CDR2 and CDR3, said CDR1, CDR2 and CDR3 consisting of the amino acid sequences of CDR1, CDR2 and CDR3 shown in the VH of the amino acid sequence of SEQ ID NO: 8; or alternatively

Wherein the monoclonal antibody comprises a VL and a VH, wherein: VL comprises CDR1, CDR2 and CDR3, said CDR1, CDR2 and CDR3 consisting of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in VL of the amino acid sequence of SEQ ID NO: 2; and, the VH comprises CDR1, CDR2 and CDR3, said CDR1, CDR2 and CDR3 consisting of the amino acid sequences of CDR1, CDR2 and CDR3 as shown in the VH of the amino acid sequence of SEQ ID NO: 4.